Use of transgenic animals in toxicology

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Use of transgenic animals in toxicology David J. Kirkland Transgenic and genetically engineered animals are being increasingly

▼ In the past 20 years, there have been phenomenal advances in the application of geneticengineering techniques. Increasingly, the whole animal is needed for biological research, pharmaceutical development and toxicological assessment.Thus, the ability to transfer functional genes to produce transgenic mice expressing foreign genes of human (or other) origin, or to inactivate genes selectively to produce knockout mice, provides a powerful tool for the advancement of the science in these key areas. Transgenic mice were initially used as models for the study of molecular biology, but they are now increasingly being applied in the wider life sciences.

been some success in modelling neurological conditions11, including complex conditions such as Alzheimer’s disease12. However, these models have hitherto not been used to study the toxicology of the various therapies that might be used to treat these diseases. That these models might be used in toxicology is of interest. Traditionally, toxicologists have tested new therapeutic agents in healthy animals, and yet the therapy is intended for diseased human patients. It has rightly been asked whether a diseased animal with, for example, an enzyme deficiency or a genetic malfunction, would exhibit greater- or less-toxic side effects to a particular new drug than a healthy animal. Hopefully, these models will be considered for such toxicological use in the future. One area where progress has been made in using transgenic animals for safety testing is vaccine testing. A transgenic model for polio virus may allow reduced use of monkeys for neurovirulence testing of vaccines, according to Nomura13. A line of mice (TgPVR21) developed by Koike et al.14, that are transgenic for the human poliovirus-receptor gene, mimic the susceptibility of human and non-human primates to poliovirus infection. Currently, significant numbers of primates are used in vaccine testing and substantial animal welfare benefits would be derived from being able to replace these with transgenic mice.

New disease models By introducing genes that are related to various diseases, it has been possible to clarify the roles of various gene functions in the onset and etiology of diseases. Noted successes have been achieved in modelling: breast1, pancreatic2 and prostate3 cancer, neurofibroma4, autosomal dominant retinitis pigmentosa5, motor-neurone death in amyotrophic lateral sclerosis6, multistep erythroleukaemias7, sickle-cell anaemia8, susceptibility to transmissible spongiform encephalopathies such as scrapie9 and diabetes10. There has even

‘Humanized’ mice As part of the current initiative to harmonize global approaches to pharmaceutical registrations, an Expert Working Group of the International Conference on Harmonisation (ICH) has been considering how to test the safety of biotechnology products. A draft guideline has been produced15 that discusses the fact that most species of animal will be inappropriate for the evaluation of the toxicity of recombinant or naturally occurring proteins designed for human therapy. This is because the animals conventionally

used in the study of diseases and for safety assessments for new products. There are four main areas in which they influence pharmaceutical development; two of these, new disease models and ‘humanized’ animals for the assessment of biopharmaceuticals, have not yet made their impact upon toxicology and so will only be briefly discussed. Models for in vivo genotoxicity testing and carcinogenicity testing, however, are already becoming established, and the advent of new harmonized guidelines for genotoxic and carcinogenic evaluation of pharmaceuticals makes it more appropriate to assess those models currently available in these areas.

David J. Kirkland Covance Laboratories Otley Road, Harrogate UK HG3 1PY tel: +44 1423 500011 fax: +44 1423 569595

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Copyright ©1998 Elsevier Science Ltd. All rights reserved. 1461-5347/98/$19.00. PII: S1461-5347(98)00016-9

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used in toxicology will most often not express the specific protein receptors or antigens with which the biopharmaceutical will react in humans. It has been suggested, therefore, that ‘humanized’ mice should be developed.This process would consist of identifying and cloning the human gene that codes for the appropriate receptor in question, making a transgenic mouse containing and expressing the gene, developing a colony of animals, and using them for toxicological testing. However, just because a transgenic mouse contains and expresses the relevant gene, this does not guarantee that the protein will be processed and function in the same way that it does in humans. Examples of transgenic models expressing relevant target proteins include:

• • • • •

The b-2-adrenergic receptor16 The interleukin-6 receptor17 The high-affinity IgE receptor18 The CD4 and major histocompatibility complex class II proteins19 Human myelin basic protein antigen20

Thus, through the production of such ‘humanized’ models, the recommendations of the ICH might be fulfilled. However, the production and breeding of enough animals for large-scale toxicological evaluation is likely to be an expensive process.There is also concern that there will be little relevant background or historical data on each ‘humanized’ model, and so the interpretation of toxicological data could be fraught with problems. Transgenic rodents in genotoxicity testing Conventional approaches to genotoxicity testing in vivo currently only allow for the detection of chromosomal damage (either as aberrations in metaphase chromosomes or as micronuclei) or DNA damage (identified as unscheduled DNA synthesis, UDS) in a limited number of tissues, usually bone marrow, blood and liver. A relatively new method, the single-cell gel-electrophoresis assay, or COMET assay21 (named after the shape produced by the DNA streaming out of each cell on the gel), allows the identification of DNA damage in any tissue from which single-cell suspensions can be made. By using a radiolabelled test chemical or 32P-postlabelling22, it is possible to determine whether any DNA adducts have been formed and, theoretically, this can be done in any animal tissue. However, none of these methods provide information as to whether the DNA adducts or damage caused have a biological consequence that would result in mutation. Therefore, a need was identified for an in vivo gene mutation system that could complement the in vitro gene mutation measurements (e.g. Ames test or mouse lymphoma tk-mutation test) that form a standard part of genotoxicity testing. In addition to

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3126 bp

lacZ cos

0

EcoR1 site 33

36

cos

47 Kb

lgt10lacZ vector Figure 1. Illustration of the lacZ construct in MutaMouse.

complementing the current battery of genotoxicity tests, such in vivo models could also be used in situations where tumours were induced in a carcinogenesis assay but conventional genotoxicity testing had produced negative results. This has now been identified as being of potential use by the ICH23. MutaMouse and Big Blue The most widely evaluated transgenic mutation models are MutaMouse24 (with a bacterial lacZ target gene) and Big Blue25,26 (with a bacterial lacI target gene). The transgenes are incorporated in a bacteriophage lgt10 vector, as illustrated in Figure 1 for lacZ. There are 40 copies of the vector in each haploid set of chromosomes in MutaMouse, located in a head-totail concatameric sequence on chromosome 3 (Ref. 27). In both of the models described, mice (and rats in the case of Big Blue) are treated with the test chemical by any route, and relevant tissues are taken and flash frozen. DNA can be extracted later, at a suitable time, and reacted with a commercial packaging mix that cuts the DNA at the cos sites surrounding the transgenic insert, allowing the pieces to be packaged into infectious bacteriophage lambda. The phage are assayed on a sensitive strain of bacteria (Escherichia coli C). In the MutaMouse system, phage carrying mutant lacZ genes can be selected out from the non-mutants by using E.coli C which is also GalE2 (lacking epimerase, and allowing the rapid build up of lethal concentrations of uridine diphosphate–galactose in non-mutant phage (Figure 2)28–30. In the Big Blue system, the conventional assay has been to identify mutant phage as those producing blue plaques on a background of clear (non-mutant) plaques25, but a new system is now available in which selection for the cII gene allows selection against non-mutant phage31. These models have been particularly useful for investigating gene mutations in tissues where it is suspected that site-ofcontact effects may occur (e.g. in skin, lung and the GI tract). Therefore, much of the validation work with MutaMouse and Big Blue has focused on a comparison between the tissues in which tumours appear and those in which significant mutation is detected. There is now a substantial database showing very good correlations for a diverse range of carcinogens. 63

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E. coli C lac- galE- Ÿ containing either lacZ + or lacZ -

Adsorption Injection

functional LacZ proteinŸ

P gal

non-functional LacZ proteinŸ Ÿ

Ÿ

Galactose

No galactose

UDP-glucose UDP-galactose

Bacterial and viral replication

metabolic activation but was very difficult to detect as an in vivo genotoxin in conventional assays. After many negative publications, MNNG was eventually reported as inducing micronuclei in bone marrow after intraperitoneal administration33. It does not induce UDS in liver, but it is clearly mutagenic in MutaMouse stomach (although not in bone marrow or liver) after oral administration and mutagenic in the skin after topical administration, thus accurately mimicking the tumour distribution32,34,35. Mutation data for skin and stomach are shown in Figure 3.

Cell death Clear plaques No plaques

Figure 2. Basis for positive selection of lacZ2 phage.

b-propiolactone b-propiolactone is a potent, direct-acting (i.e. it does not require metabolic activation) in vitro mutagen and a site-ofcontact carcinogen, but it is negative for induction of micronuclei in bone marrow or peripheral blood and does not induce UDS in mouse liver when administered orally (the normal route for this test). However, it does induce micronuclei in liver and testes when given intraperitoneally. Brault et al.32 showed that, when tested in MutaMouse, significant mutations were induced in stomach (one of the sites of tumour induction) but not in bone marrow or liver. N-methyl-N´-nitro-N-nitrosoguanidine Another compound that is a site-of-contact carcinogen (i.e. for the skin when applied topically and for the stomach when administered orally) is N-methyl-N´-nitro-N-nitrosoguanidine (MNNG). This is a potent in vitro mutagen that does not need

Mutants per 106 pfu

2,500 2,000 1,500 1,000 500 0

0

50

100

0

250

500

Single oral dose (mg kg-1). Topical dose (mg) Figure 3. Induction of lacZ mutations in MutaMouse stomach and skin by N-methyl-N'-nitro-N-nitrosoguanidine. ■ stomach three days, ■ stomach ten days, ❑ skin seven days, ■ skin 21 days.

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4-Nitroquinoline-N-oxide 4-Nitroquinoline-N-oxide (NQO) is carcinogenic at a number of sites following oral administration, but the prime sites for tumours are the glandular stomach and the lungs. NQO is readily detected as a genotoxin in vivo, inducing micronuclei in bone marrow but not UDS in liver.The relative mutagenic activity of NQO in various tissues of MutaMouse is, however, a remarkable parallel of its carcinogenic activity. Coates and Dean36 have shown that NQO is a potent mutagen for the mouse stomach, less mutagenic in marrow, liver and lung, and weakly mutagenic in the testes (Figure 4). Dimethylnitrosamine Dimethylnitrosamine is an in vitro mutagen that is more potent in the presence of rat liver metabolic activation than in its absence. It has been difficult to demonstrate the induction of micronuclei in bone marrow in vivo, although it readily induces micronuclei and UDS in liver. It is generally recognized as a potent liver carcinogen, also producing tumours of blood vessels and the lungs but not of the stomach, where it is also unable to induce UDS. After prolonged dietary treatment (105 days), mutation was detected clearly in the liver but not in the forestomach of transgenic mice37,38. Chloromethylpyrene Chloromethylpyrene (CMP) is an interesting compound because it is a mutagen in vitro after metabolic activation, produces micronuclei in liver but not bone marrow, and yet is a probable skin carcinogen. When tested in MutaMouse, it was clearly mutagenic in skin after topical administration, but was not mutagenic in stomach after oral administration34 (Figure 5). The mutagenic profile suggests that CMP might be a skin carcinogen but it is unlikely to be a stomach carcinogen after oral administration, although the micronucleus data suggest that it might prove to be a liver carcinogen. Other compounds Other compounds that have shown organotropic effects are the food mutagen 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ), the skin carcinogen 7,12-dimethylbenz(a)anthracen and the ethylating agents ethylnitrosourea and diethylnitrosamine.

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3,000

Stomach

Marrow

Liver

Lung

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Mutation frequency x 10-6

2,500

2,000

1,500

1,000

500

0 7

14

28

7

14

28

7

14

28

Days after single oral dose of 200 mg

7

14

28

7

14

28

kg-1

Figure 4. Induction of lacZ mutations in various tissues of MutaMouse by 4-nitroquinoline-N-oxide. ■ control, ❑ NQO.

There are very few reported studies on known non-carcinogens in either MutaMouse or Big Blue, and so the specificity (i.e. accuracy of giving negative results with inactive chemicals) of these models is not clear. The tissue-specific effects mentioned above, in which some organs clearly did not exhibit any mutagenic response, suggest that the cells of these transgenic mutation models are not so exquisitely sensitive that they will produce a positive response to any insult. It has taken many years for sufficient experiments to be reported with these transgenic mutation models, but there now appears to be a role for them in safety testing. For some time there was concern that multiple expression times would be

Short- and medium-term tumour models The ICH carcinogenicity working group recently recommended that, for pharmaceuticals, it might be possible to conduct only one lifespan bioassay, accompanying this with a short- or medium-term tumour assay40.The choices are:

250 Mutants per 106 pfu

needed to ensure that positive responses would not be missed. However, it now appears that, although some mutations arise quite quickly, they are then reasonably persistent; others might take a few weeks to appear. As suggested by Heddle et al.39 it is possible to envisage a protocol in which animals are dosed over a five-day period and sampled after a further 28 days. From all the data we have seen, including the examples above, the majority of carcinogens would be detected by this protocol. Thus, the transgenic mutation models offer considerable advantages in following up on in vitro genotoxicity positives, in predicting tumour induction and in helping to resolve genotoxic and non-genotoxic carcinogens.

200 150

• • •

100 50

Initiation promotion model Neonatal mouse Transgenic (or genetically engineered) mouse models

0 0

25

50

0

5

10

Single oral dose (mg kg-1). Topical dose (mg) Figure 5. Induction of lacZ mutations in MutaMouse skin after topical application but not in stomach after oral dosing with chloromethylpyrene. ■ stomach three days, ■ stomach ten days, ❑ skin seven days, ■ skin 21 days.

The initiation promotion model is not a transgenic model but is worthy of consideration in this context. It was originally developed with the liver as the target organ41. Diethylnitrosamine is used as the initiator, followed two weeks later by a six-week post-treatment with the test drug, and tumour sites are scored as glutathione-S-transferase placental form (GST-P)-positive foci.The results for 206 compounds are summarized in Table 1. Genotoxic 65

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Table 1. Results from initiation/promotion model [number of results/number of chemicals (%)] Carcinogenicity

Ames+

Ames–

Ames?

Hepatocarcinogens 23/24 (96)a 22/26 (85)b 1/1 (100) Non-hepatocarcinogens 6/22 (27) 2/13 (15) 0/2 (0) Non-carcinogens 0/6 (0) 1/30 (3)c 0/2 (0) Unknown 3/13 (23) 16/50 (32)d 3/16 (18) Total 36/65 (49) 42/120 (35) 4/21 (19) a4,4¢-diaminophenylmethane

Total 46/51 (90) 8/37 (22) 1/38 (3) 23/80 (29) 78/206 (38)

was negative. bClofibrate, di-(2-ethylhexyl) adipate, di-(2-

ethylhexyl) phthalate and trichloroacetic acid (peroxisome proliferators) were negative. cMalathion

was positive. dFour equivocal carcinogens were negative and one was positive.

and non-genotoxic hepatocarcinogens are detected efficiently, although four carcinogenic peroxisome proliferators gave negative results.The model is now being extended as a multi-organ bioassay42 in which five different initiating chemicals are administered over a four-week period, and post-treatment with the test chemical (e.g. in diet) is performed over a further 24 weeks. Very few data have been published so far.The investigation of low doses of some heterocyclic amines that are carcinogenic alone in the intestine of mice revealed some synergistic induction of intestinal adenocarcinomas with the initiators. However, there are concerns that data from this model might be difficult to interpret because of potential interactions between so many initiators. The neonatal mouse is also not a transgenic assay, but it is important to evaluate its performance in comparison with the transgenic tumour models (see later). In this assay, newborn mice are dosed with the drug on two or three occasions during the first two weeks of life and the animals are observed over one year for the appearance of tumours. It has been usual to dose via intraperitoneal or subcutaneous routes, but gavage dosing is possible. The most extensive published database43 (45 compounds, although only 37 had been tested in conventional carcinogenicity assays; summarized in Table 2) revealed very good detection (21/23) of trans-species carcinogens, but a lack of sensitivity Table 2. Results from neonatal mouse assay

Trans-species Mcarcinogens Single-species Mcarcinogensc Non-carcinogens a3-OH-anthranilic

No. of chemicals

Negative in neonatal

Positive in neonatal

23

2a

21

5

4b

1

9

9

0

acid, phenobarbital. b4-OH-4-acetylaminobiphenyl, oestradiol, 3-OH-

kynurenine, isonicotinic acid hydrazide. cTwo were tested only in a single species.

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(1/5) for single-species carcinogens, which can often be nongenotoxic in mechanism and of less biological relevance. This is thought to be because of the fact that, at the time of treatment, neonates experience a phase of maximum cell proliferation, and so the effects of additional cell proliferation induced by the test chemical will be negligible. Some scientists prefer this model to the other alternatives because the animals are not genetically altered and the dosing and observation schemes combine to reduce the chances of detecting non-genotoxic carcinogens. There are four short- or medium-term transgenic models being evaluated. The genetic changes that are relevant to the carcinogenic process are:

• • •

Activated oncogene (Tg.AC model,TgHras2 model) Inactivated tumour suppressor gene (p531/2 model) Inactivated DNA-repair gene (XPA2/2 model)

Tg.AC model In these animals, four copies of the v-Ha-ras oncogene are located in tandem on chromosome 11 of strain FVB/N mice. There are hemizygous and homozygous versions, but the latter appear to respond more consistently. Mice (males in particular) are usually housed singly because scratches from fighting can lead to the occurrence of spontaneous skin papillomas. Animals are usually exposed dermally (although the oral route is being explored) for 20 weeks and observed for a further six weeks for the appearance of skin papillomas.As can be seen from Table 3, the model is good at detecting mutagenic and non-mutagenic carcinogens, including tumour promoters, but fails to detect ethyl acrylate and Nmethyl-o-acrylamide. Ethyl acrylate requires cell proliferation to function as a stomach carcinogen.With dermal dosing as the current routine in this model, it might, therefore, fail to detect certain tissue-specific carcinogens of this type. However, further evaluation of this model will examine the effects of oral dosing. There is also concern that the model may be oversensitive in other ways, in that it gave positive results with two out of five nonmutagenic non-carcinogens (resorcinol and rotenone). Some of these results have been published by Tennant and coworkers44,45; other results are available in the US NIEHS/NTP preliminary database, but remain unpublished in a peer-reviewed journal. p53+/2 model In this model, one allele of the gene encoding p53 of C57Bl/6 mice has been selectively inactivated to produce an animal that is analogous to humans at risk from heritable forms of cancer (e.g. Li–Fraumeni syndrome). Mice can be dosed by any appropriate route over a 26-week period. As can be seen from Table 4, the model is good at detecting mutagenic carcinogens (including benzene and phenolphthalein, which are clastogens rather than gene mutagens) but does not detect non-mutagenic carcinogens

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Table 3. Results from Tg.AC model Chemical group

No. tested Positive Negative results results

Mutagenic carcinogens Non-mutagenic carcinogens Mutagenic non-carcinogens Non-mutagenic non-carcinogens

7 7 3 5

aEthyl

7 5 2b

2a 3 3

acrylate, N-methyl-o-acrylamide. bResorcinol, rotenone.

or non-carcinogens.Therefore, it behaves rather like a long-term genotoxicity assay and, for those wanting a model which is like the conventional mouse life-span bioassay, and will detect nongenotoxic carcinogens, it is insensitive. However, because there are no examples of mouse-only carcinogens being regulated (according to the ICH) and, therefore, the carcinogens that concern humans are those that are trans-species, this model, in conjunction with a normal rat life-span bioassay, could help to eliminate those mouse-only, non-genotoxic positives that have caused so many problems for so many pharmaceutical companies in the past. Results with some chemicals in this model have also been published by Tennant and co-workers44,45 and other data can be found in the US NIEHS/NTP preliminary database. TgHras2 model In this model strain CB6F1 mice carry five or six copies per cell of the human c-Ha-ras oncogene with its own promoter. Again, animals can be dosed by any appropriate route over a period of 26 weeks. As can be seen from Table 5, the model is good at detecting mutagenic carcinogens, and also transspecies non-mutagenic carcinogens. It did not detect the mouse-only non-mutagenic carcinogen 1,1,2-trichloroethane, nor three out of three non-mutagenic non-carcinogens. There is some concern over the occurrence of spontaneous tumours in control mice after approximately 30 weeks (forestomach papillomas, spleen haemangiomas, lung adenocarcinomas, skin tumours and lymphomas) and whether these could occur in chemically-treated animals. For example, an equivocal Table 4. Results from

p53+/–

No. tested Positive Negative results results

Mutagenic carcinogens Non-mutagenic carcinogens Mutagenic non-carcinogens Non-mutagenic non-carcinogens

6 6 2 3

aIncludes

response (i.e. increase in spontaneous tumours) was seen with the mutagenic non-carcinogen 4-nitro-o-phenylenediamine, and it is not clear whether this response is a true or false positive. Data on some of these chemicals have been published by Yamamoto et al.46,47 but other results are as yet unpublished. XPA2/2 model In this model, C57Bl/6 mice have a deletion across exons 3 and 4 of both XPA alleles, rendering the cells totally defective in nucleotide excision repair48. Thus, the animals are comparable with human repair-deficient cancer-prone conditions such as Xeroderma pigmentosum: the mice are sensitive to tumour induction by UV-B49, but have also given positive responses with four other mutagenic carcinogens, as can be seen from Table 6. Some of the data have been published50 but, as yet, most are unpublished. There are no other data available at present to indicate how specific this model is (will it detect non-mutagenic carcinogens such as the Tg.AC and TgHras2, or only detect mutagenic carcinogens, as the p53+/2 does?). Concern has rightly been expressed that, because the conventional mouse carcinogenicity bioassay has frequently produced results that were considered to be irrelevant for humans (and, hence, the lack of regulatory action on mouse-only carcinogens), the results from the new mouse models might not be any better. Of course, these models save on animals (120–150 per compound instead of 500–600 in a conventional mouse bioassay) and, therefore, space and resources, but because they are in addition to a conventional bioassay in, for example, the rat, the overall development time for a drug is not shortened.Thus, it is important to know that the predictability for human carcinogenicity is improved by transferring to these new models. Further evaluation of these four transgenic mouse models, and the neonatal mouse model, is under way in a large collaborative study sponsored by the International Life Sciences Institute of the US Health and Environmental Sciences Institute. A total of 21 chemicals are being tested, including: Table 5. Results from TgHras2 model Chemical group

No. tested Positive Negative results results

Mutagenic carcinogens Non-mutagenic carcinogens M(trans-species) Non-mutagenic carcinogens M(mouse only) Mutagenic non-carcinogens Non-mutagenic non-carcinogens

17 5

model

Chemical group

6a 6 2 3

benzene & phenolphthalein which are Ames-negative but micronucleus-positive.

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17 5 1a

1 3 3

1b

2 3

a1,1,2-trichloroethane. b4-nitro-o-phenylenediamine.

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Derived Pharmaceuticals. International Conference on Harmonisation of

Table 6. Results from XPA–/– model Compounds/ agents XPA–/–: UV–B DMBA BP 2-AAF PhiP

Ames result

+ + + + +

Target organs in non-transgenics

skin skin multiple bladder, liver lymphoma

Technical Requirements for Registration of Pharmaceuticals for Human

XPA–/–

XPA–/–

route

result

dermal skin paint gavage feed feed

+ + + + +?

XPA–/–/p53+/–: BP

+

multiple

gavage

Use, Step 4 document, ICH4 meeting, Brussels 16

Andre, C. et al. (1996) Eur. J. Biochem. 241, 417–424

17

Udagawa, N. et al. (1995) J. Exp. Med. 182, 1461–1468

18

Dombrowicz, D. et al. (1996) J. Immunol. 157, 1645–1651

19

Yeung, R.S. et al. (1996) Eur. J. Immunol. 26, 1074–1082

20

Altmann, D.M. et al. (1995) J. Exp. Med. 181, 867–875

21

Tice, R.R., Andrews, P.W. and Singh, N.P. (1990) DNA Damage and Repair in Human Tissues (Sutherland, B.M. and Woodhead, A.D., eds), pp. 291–301, Plenum Press

22

Randerath, K., Reddy, M.V. and Gupta, R.C. (1981) Proc. Natl.Acad. Sci. U. S.A.

23

ICH (1997) Topic S2B: Genotoxicity: A Standard Battery for Genotoxicity

78, 6126–6129

+

Testing of Pharmaceuticals. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human

• • • •

Human genotoxic and non-genotoxic carcinogens Hormones Rodent carcinogens that are thought not to be human carcinogens Non-carcinogens

In the light of the comments above, it is significant that the emphasis is on ‘evaluation’ rather than ‘validation’. In other words, the aim is not to be able to detect rodent carcinogens but to improve the process of human risk assessment. Let us hope these laudable objectives are achieved.

Use, Step 4 document, ICH4 meeting, Brussels 24

Gossen, J.A. et al. (1989) Proc. Natl.Acad. Sci. U. S.A. 86, 7971–7975

25

Kohler, S.W. et al. (1991) Proc. Natl.Acad. Sci. U. S.A. 88, 7958–7962

26

Kohler, S.W. et al. (1991) Environ. Mol. Mutagen. 18, 316–321

27

Swiger, R.R., Myhr, B. and Tucker, J.D. (1994) Mutat. Res. 325, 145–148

28

Gossen, J.A. et al. (1992) Nucleic Acids Res. 20, 3254

29

Dean, S.W. and Myhr, B. (1994) Mutagenesis 9, 183–185

30

Brooks,T.M. et al. (1995) Mutagenesis 10, 149–150

31

Jakubczak, J.L. et al. (1996) Proc. Natl.Acad. Sci. U. S.A. 93, 9073–9078

32

Brault, D. et al. (1996) Mutat. Res. 360, 83–87

33

Ashby, J. and Mirkova, E. (1987) Mutagenesis 2, 199–204

34

Brooks,T.M. and Dean, S.W. (1996) Mutagenesis 11, 529–532

Acknowledgements I wish to thank Steve Dean for helpful advice and criticism during the compilation and review of this manuscript.

35

Myhr, B.C. (1991) Environ. Mol. Mutagen.18, 308–315

36

Coates, A. and Dean, S.W. Mutagenesis (in press)

37

Shephard, S.E., Gunz, D. and Schlatter, C. (1995) Food Chem.Toxicol. 33,

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