Mutagenicity of 4-nitroquinoline 1-oxide in the MutaMouse.

As part of a collaborative study, the Mammalian Mutagenesis Study Group (MMS), a sub-organization of the Environmental Mutagen Society of Japan (JEMS) conducted mutagenicity tests in MutaMouse. Using a positive selection method, we studied the organ-specificity and time dependence of mutation induction by 4-nitroquinoline 1-oxide (4NQO). A single dose of 4NQO was administered intraperitoneally (7.5 or 15 mg/kg) or orally (200 mg/kg) to groups of male mice. On days 7, 14 and 28 after treatment, we isolated the liver, kidney, lung, spleen, bone marrow, testis and stomach in the intraperitoneal administration experiment and the liver, lung, bone marrow, testis and stomach in the oral administration experiment. In addition, we performed the peripheral blood micronucleus test to evaluate clastogenicity. In the mice treated intraperitoneally at 7.5 mg/kg, we found increased mutant frequency (MF) only in the lung, where the MF did not vary with expression time. In the mice treated at 15 mg/kg, we found increased MF in the liver, bone marrow and lung. In orally treated mice, the MF was high in the lung and liver and very high in the bone marrow and stomach while the increase in the testis was negligible. As the expression time was prolonged, the MF tended to increase in the liver, decrease in the bone marrow, and remain stable in the lung, testis and stomach. The incidence of micronucleus induction in peripheral blood cells was significantly increased (p<0.01) in the 4NQO groups when compared with the vehicle control group by intraperitoneal treatment. Thus, these assay systems appeared to be of use in detecting not only genetic mutation but also chromosomal aberration.

Transgenic mouse mutation assay systems can play an important role in regulatory mutagenicity testing in vivo for the detection of site-of-contact mutagens.

Transgenic mouse mutation assays, such as MutaMouse (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 MutaMouse 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.

Environmental enrichment of laboratory animals used in regulatory toxicology studies.

There is a wealth of information in the published literature which describes a multitude of approaches to enriching the environment of laboratory animals. This paper attempts to review the various methods of enrichment through social contact, enhancement of the environment and diet, and improvements in husbandry. It attempts to place the various enrichment initiatives within the context of a laboratory which conducts regulatory toxicology, describes some of the experiences in the author's own laboratory and attempts to highlight those ideas which might prove practical to implement in the future. The aim is to demonstrate that a creative approach to environmental enrichment is indeed compatible with regulatory toxicology. It is hoped that this will encourage those responsible for the care and welfare of animals in such a laboratory to challenge historical practices and include environmental enrichment as a fundamental necessity of study design.

Benzo[a]pyrene site of contact mutagenicity in skin of Muta Mouse.

Benzo[a]pyrene (BP) has been investigated for the ability to induce mutation at the site of contact. Skin painting treatments with BP caused a time-dependent and statistically significant increase in mutation frequency (MF) in the treated areas of skin. The MF exceeded 500 x 10(-6) 21 days after either 1 x 25 or 5 x 5 micrograms treatments. Increases to > 700 x 10(-6) were seen when doses of 1 x 50 or 5 x 10 micrograms were used. Neither the liver nor the lung showed any increase in mutation frequency after 21 days in animals exposed to the 5 x 10 micrograms treatment regime. It is concluded that following topical administration, BP is able to induce mutation in the skin at the site of application, but not in either the lung or liver.

The detection of gene mutation in the tubular sperm of Muta Mice following a single intraperitoneal treatment with methyl methanesulphonate or ethylnitrosourea.

Transgenic mouse assays, such as Muta Mouse, provide a method to predict the potential target organ carcinogenicity of chemical compounds. As part of a collaborative study, the effects of the direct-acting mutagens, methyl methanesulphonate (MMS) and ethylnitrosourea (ENU), were investigated for gene mutation in the tubular sperm of Muta Mice testes after a single intraperitoneal exposure. Groups of male Muta Mice were dosed intraperitoneally with either 1/15 M phosphate buffer, pH 6.0 (vehicle control), 40 mg/kg methyl methanesulphonate (MMS) or 150 mg/kg ethylnitrosourea (ENU). The animals were sacrificed 14 days after the single dose. Mutation frequencies were determined in tubular sperm DNA. The results showed a mean mutation frequency (MF) of 2.1 x 10(5) (64 mutants per 3.05 x 10(6) PFU) for the 10 vehicle-treated mice, a mean MF of 2.8 x 10(5) (78 mutants per 2.75 x 10(6) PFU) for the 10 MMS-treated mice and a mean MF of 9.1 x 10(5) (194 mutants per 2.14 x 10(6) PFU) for the 8 ENU-treated mice; this latter value representing a 4.5-fold increase over the vehicle control values.

Detection of gene mutation in skin, stomach and liver of MutaMouse following oral or topical treatment with N-methyl-N'-nitro-N-nitrosoguanidine or 1-chloromethylpyrene: some preliminary observations.

Transgenic mouse assays, such as MutaMouse, provide a method to predict the potential target organ carcinogenicity of chemical compounds. As part of a validation study, the effects of the direct-acting mutagens, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 1-chloromethyl-pyrene (CMP), were investigated for gene mutation in the tissues of MutaMice after a single oral or topical exposure. MNNG (50 or 100 mg/kg) or CMP (25 or 50 mg/kg) were administered as a single oral dose and the mice killed after 3, 7 or 10 days. Mutation frequencies were determined in stomach DNA from both MNNG and CMP-treated animals and in liver DNA from the MNNG-treated animals only. The results, although obtained from a limited number of animals, consistently showed that MNNG increased the mutation frequency in stomach DNA, but not apparently in liver DNA, at each exposure time; no clear increase in mutation frequency was seen in the stomach DNA of CMP-treated animals. Also, MNNG (250 or 500 micrograms) or CMP (5 or 10 micrograms) in acetone were applied as a single dose to the shorn skin of mice 7, 14 or 21 days prior to death. A positive control group was similarly given dimethyl-benz[a]anthracene (DMBA, 40 micrograms) and sacrificed after 14 days. Mutation frequencies were determined in the skin DNA extracted from all animals and in the stomach DNA from MNNG-painted animals only. The results, again obtained from a limited number of animals, clearly showed that all test compounds consistently increased the mutation frequency of skin DNA and that these increases were far greater in the DMBA- and MNNG-treated mice than the CMP-treated mice. No apparent increases were seen in the stomach DNA from the MNNG-painted mice.

The detection of gene mutation in transgenic mice (Muta Mouse) following a single oral dose of 2-acetylaminofluorene.

Transgenic mouse assays, such as Muta Mouse, provide a method to predict the potential target organ carcinogenicity of chemical compounds. As part of a validation study, 2-acetylaminofluorene was administered as a single oral dose of either 50 or 100 mg/kg. The mice were killed 3, 7, 14, 28, 56 or 112 days after this single treatment. Mutation frequencies were determined in liver DNA. The results showed that a mutagenic response was observed in the mice treated at the highest dose (100 mg/kg) and this increase was expressed from 28 up to 112 days after the single exposure.

On the need for confirmation of negative genotoxicity results in vitro and on the usefulness of mammalian cell mutation tests in a core battery: experiences of a contract research laboratory.

There are currently unresolved discussions on two important topics in regulatory genetic toxicology, namely whether or not it is necessary to confirm clearly negative results from in vitro assays in independent experiments, and whether or not the mammalian cell gene mutation test should be part of a core battery of tests. Analysis of in-house data, using full regulatory protocols, suggests that for bacterial mutation tests (144 compounds reviewed) it is impractical to design a single experiment to incorporate all relevant variables and, therefore, confirmation of negative results using modified methodology is desirable. On the other hand, data from TK mutation assays (65 compounds reviewed) and chromosomal aberration tests (94 compounds reviewed) suggest that confirmation of negative results in well-designed mammalian cell studies is not necessary. Analysis of 32 chemicals, each tested in Ames, TK mutation and chromosomal aberration tests, revealed two positives unique to the TK assay and one unique to the chromosomal aberration test. As the TK assay did not show increased susceptibility to false positives (frequency of positives is similar to other in vitro assays) and these two unique positives were clearly observed (> 2-fold increase in mutation frequency at 60-70% relative survival in both cases), they do appear to be 'real' results. Both compounds induced small colony mutants (one also induced 'large'), and yet in vitro chromosomal aberration and in vivo micronucleus tests were negative. The single unique chromosomal aberration positive may be an artefact of high cytotoxicity, and certainly the substance was negative for micronuclei and UDS in vivo, so it might be argued that the chromosomal aberration test is surplus to requirements. Overall, however, it would seem premature to reject either assay at this time, and experience suggests the extra information provided by two mammalian cell tests instead of one is extremely valuable in assessing risk and deciding upon appropriate follow-up tests in vivo.

Genotoxicity studies with chloroethane.

In a recent 2-year inhalation study with F344 rats and B6C3F1 mice conducted as part of the U.S. National Toxicology Program (NTP, 1989), chloroethane (ECl) at an exposure concentration of 15,000 ppm induced a high incidence of endometrial uterine carcinomas only in female mice but not in rats, leading to the conclusion of "clear evidence of carcinogenicity" for the mouse. In order to elucidate whether a genotoxic effect may be a critical factor for the carcinogenicity of ECl in the mouse, we have performed three genotoxicity tests: (1) in vitro HPRT test with CHO cells according to a specially developed gas protocol, (2) in vivo/in vitro UDS with female B6C3F1 mice at an exposure concentration of 25,000 ppm (6 h/day, 3 days), (3) in vivo micronucleus assay with male and female B6C3F1 mice exposed to 25,000 ppm ECl according to the same schedule. In the in vitro HPRT test a mutagenic potential of ECl was detected in the presence as well as in the absence of S9 mix. In contrast, both in vivo test systems failed to detect any indications of genotoxicity of chloroethane at an exposure concentration even higher than that of the NTP study. It is suggested that in vivo the genotoxic potential of ECl is so low that an assumed genotoxic damage is below the detection limit of the test systems used. This leads to the conclusion that genotoxicity may not be a key factor in the induction of the uterine carcinomas in the B6C3F1 mouse.

Recommendations for the performance of UDS tests in vitro and in vivo.

The Working Group (WG) dealt with the harmonization of routine methodologies of tests for unscheduled DNA synthesis (UDS) both in vitro and in vivo. In contrast to the existing guidelines from OECD, EPA and EC on in vitro UDS tests (there is no Japanese UDS guideline), the Working Group recommends that in general in vitro UDS tests should be performed with primary hepatocytes. For routine applications any other cell types would need special justification. Hepatocytes from male rats are preferable, unless there are contra-indications on the basis of e.g. toxicokinetic data. According to the OECD, EPA and EC guidelines, UDS may be analysed by means of autoradiography (AR) or liquid scintillation counting (LSC). The WG recommends use of AR. LSC is less suitable due to the problem of differentiation between UDS activity and replicative DNA synthesis, and the disadvantage that cells cannot be analysed individually. Since a specific cell type was recommended by the WG, methodological aspects could be described in more detail than in the present guidelines. For in vitro tests, it was agreed that the initial viability of freshly isolated hepatocytes should be at least 70%. With regard to the need for confirmatory experiments in the event of a clear-cut negative result, the majority view was that confirmation by a second (normally not identical) experiment is still needed; this is in line with the present OECD and EC guidelines. Evaluation of results from UDS tests should be based primarily on net nuclear grain (NNG) values, although it is recognised that nuclear and cytoplasmic grains result from different biological processes. Since grain counts are influenced by a number of methodological parameters, no global threshold NNG value can be recommended for discrimination of positive and negative UDS results. For in vitro assays, the criteria for positive findings go beyond those of the present guidelines and two alternative approaches are given which are based on (1) dose-dependent increases in NNG values and (2) reproducibility, dose-effect relationship and cytotoxicity. At present there is no official guideline on the performance of in vivo UDS tests. Some fundamental recommendations given for in vitro methodology also apply to the in vivo assay. For routine testing with the in vivo UDS test, again the general use of hepatocytes from male rats is recommended. However, concerning the requirement to use one or two sexes, consistency with other in vivo genotoxicity assays (e.g. the micronucleus assay) would be preferable. As for the in vitro methodology, AR is preferred rather than LSC.(ABSTRACT TRUNCATED AT 400 WORDS)

Measurement of gene mutation in vivo using Muta Mouse and positive selection for lacZ- phage.

The use of transgenic animals for in vivo mutagenicity studies following rescue of the bacterial lacZ transgene has been hindered by the sheer scale of the experimental work involved. We describe here a new positive selection protocol which is based upon a modified E. coli bacterial host. This new system is potentially capable of generating mutation data much faster and more cheaply than previous methods.

Development of assays for the detection of photomutagenicity of chemicals during exposure to UV light. II. Results of testing three sunscreen ingredients.

Three sunscreen ingredients, derivatives of benzylidene camphor, were tested for photomutagenic potential. These were benzenesulfonic acid, 4-[(4,7,7,-trimethyl-3-oxo-bicyclo [2.2.1] hept-2-ylidene) methyl] (Mexoryl SL), 4-(2-oxo 3-bornylidenemethyl) phenyl trimethylammonium methyl sulphate (Mexoryl SO) and 3,3'-(1,4-phenylenedimethylidyne) bis [7,7-dimethyl-2-oxo-bicyclo [2.2.1] heptane-1-methanesulfonic acid] (Mexoryl SX). Two complementary assay systems were used, one involving the induction of reverse mutations in Escherichia coli strain WP2, the other measuring the induction of chromosome damage in Chinese hamster ovary (CHO) cells. Irradiation with UVA and/or UVB was provided by an Osram Ultra-Vitalux sunlamp. None of the three sunscreens, tested either to the limit of solubility or toxicity, gave any indication of photomutagenicity in either assay, under conditions in which the positive control compound, 8-methoxypsoralen, was extremely photomutagenic. It is concluded that Mexoryls SL, SO and SX can be exposed to UV light without producing photomutagenicity measurable using a bacterial reverse mutation or a mammalian chromosome aberration assay.

Analysis and quantification of a mixed exo-acting and endo-acting polysaccharide depolymerization system.

A new mathematical model has been proposed based on a model presented by Suga, van Dedem, and Moo-Young. The model requires a separate differential equation for each polymeric species (differentiated by degree of polymerization) in the reaction mixture. The main contribution of this model is the incorporation of experimental molecular weight distributions as the initial conditions. These molecular weight distributional as the initial conditions were obtained using modern analytical equipment previouly unknown for this application. The equipment, SEC/LALLS, measures relative concentrations of specific molecular weight species along with the corresponding molecular weights, thus yielding (through some mathematical manipulation) the absolute concentration of each molecular weight species. The concentration at each molecular weight can then be incorporated as the initial condition for that equation. Theoretically, the system of differential equations can be solved to give a more realistic time course of reaction.Synergism between endo-acting and exo-acting enzymes was examined theoretically using the mathematical model. Through model predictions, it was found that synergy is based on two fundamental parameters: (1) each enzyme's activity relative to the sum of enzyme activities and, (2) overall substrate concentration relative to the exo-acting enzyme's Michaeiis kinetic constant K(m). Theoretically, synergism increases as a function of reaction time. Intermediate endo fractions (ratio of endo-acting enzyme activity to the sum of endo-acting and exo-acting enzyme activity) from 0.3 to 0.7 exhibit the most synergism. Values of kappa [log(K(m, exo)/S(0))] above about zero also exhibits the most synergism.An examination of experimental data obtained both by SEC/LALLS and by reducing sugar measurements shows that the model is inadequate for successfully predicting quantities associated with the substrate during reaction. This is especially true for synergism predictions. At short reaction times, the model predicts the data fairly well, but at longer times the predictions are inconsistent with experimental data. These inconsistencies may be due to complicating phenomena such as enzyme inhibitions.

Development of assays for the detection of photomutagenicity of chemicals during exposure to UV light--I. Assay development.

Two complementary assay systems have been adapted for the detection of compounds which may form mutagenic photoproducts during exposure to UV light from an Osram Ultra-Vitalux sunlamp as used in the evaluation of the effectiveness of sun filters. The effects of UVA and of UVB were evaluated. A bacterial plate test using Escherichia coli strain WP2 allowed the bacteria, co-plated with test chemical in soft agar, to be irradiated with various doses of UV light. Mutagenesis was assessed by scoring numbers of tryptophan-independent colonies. The chosen reference compound was 8-methoxypsoralen (8-MOP) which was non-mutagenic alone at the highest dose tested (1000 micrograms/plate). Following simultaneous exposure of bacteria to 8-MOP and doses of UV light which alone had little effect, large numbers of revertants were scored. Numbers of mutants were dependent upon the doses of both 8-MOP and of UV light. The second test system involved the exposure of Chinese hamster ovary cells to UV light in the presence of test chemical to determine the clastogenic effects of photoproducts. Treatment with 8-MOP alone up to 50 micrograms/ml was not clastogenic but concomitant exposure to non-damaging doses of UV light caused large increases in the incidence of chromosome aberrations of all types. Damage was again dependent on the doses of both components. Two additional photoactive compounds, para-aminobenzoic acid and chlorpromazine both show photoclastogenic but not photomutagenic properties. These two complementary assay systems take advantage of using no-effect levels of UV light as a baseline against which photomutagenicity readily can be compared.(ABSTRACT TRUNCATED AT 250 WORDS)

Instability of extrachromosomal cosmid DNA in SV40-transformed human (ataxia-telangiectasia) cells.

The ability of SV40-transformed human (ataxia-telangiectasia) fibroblasts to maintain Epstein-Barr virus (EBV)-based plasmids and cosmids extrachromosomally has been investigated. Transfection of a culture of cells with two different plasmids gave rise to cell clones which were able to maintain both plasmids extrachromosomally. When an EBV-based cosmid library was transfected into the cells and an individual cell clone was isolated, the extrachromosomal DNA derived from the cosmid contained numerous deletions and rearrangements. When individual cosmids were transfected into the culture, and several cell clones were isolated, the intracellular cosmid-derived DNA again showed the presence of multiple deletions and rearrangements. We conclude that although SV40-transformed cells are able to maintain more than one different EBV-based plasmid extrachromosomally, large EBV-derived molecules are extensively rearranged. SV40-transformed human fibroblasts cannot therefore be usefully used in attempting to clone genes from EBV-based cosmid libraries.

Repair of 8-methoxypsoralen + UVA-induced damage in specific sequences in chromosomal and episomal DNA in human cells.

A study of the repair of DNA damage in the dihydrofolate reductase (dhfr) gene of SV40-transformed human fibroblasts after treatment with 8-methoxypsoralen (8MOP) and UVA is described. 8MOP+UVA-induced cross-links in the dhfr gene were completely repaired by 12 h in one normal and one Fanconi's anaemia (FA) group A cell line. In contrast, approximately 35% of cross-links in an episomally maintained Epstein--Barr virus derived plasmid remained unrepaired even after 48 h. Cross-linkable monoadducts in the dhfr gene were repaired more slowly than cross-links, and there was no detectable repair of cross-linkable monoadducts in the plasmid. Thus the ability of a cell to repair 8MOP+UVA-induced cross-links or cross-linkable monoadducts in an episome does not reflect its capacity to repair such lesions in genomic DNA.

Inactivation by nitrogen mustard of plasmids introduced into normal and Fanconi's anaemia cells.

An SV40-transformed Fanconi's anaemia (FA) cell line, GM6914, exhibits approximately 2.4-fold increased sensitivity to the cytotoxic effects of nitrogen mustard (NM) when compared with the normal line, MRC5-V1. Host cell reactivation of NM-treated plasmid has been investigated using transient expression vectors which contain the chloramphenicol acetyltransferase (CAT) gene. In both cell types there is a similar, dose-dependent reduction in CAT expression which correlates with an increase in NM-induced DNA-interstrand crosslinking. The data are consistent with two possible mechanisms for inactivation of the plasmid. Either a single crosslink anywhere within the plasmid is sufficient to prevent transcription of the cat gene. Alternatively, inactivation may result from some other more prevalent NM-induced lesions within the cat coding sequence.