Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test

As part of a larger literature study on transgenic animals in mutagenicity testing, test results from the transgenic mutagenicity assays (lacI model; commercially available as the Big Blue(R) mouse, and the lacZ model; commercially available as the Mutatrade markMouse), were compared with the results on the same substances in the more traditional mouse bone marrow micronucleus test. 39 substances were found which had been tested in the micronucleus assay and in the above transgenic mouse systems. Although, the transgenic animal mutation assay is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation, the results for the majority of substances were in agreement. Both test systems, the transgenic mouse assay and the mouse bone marrow micronucleus test, have advantages and they complement each other. However, the transgenic animal assay has some distinct advantages over the micronucleus test: it is not restricted to one target organ and detects systemic as well as local mutagenic effects.     

Salmonella typhimurium as a test system for detecting the mutagenic activity of environmental pollutants. I. The mutagenic action of heavy metal salts in in vivo and in vitro systems without metabolic activation]

The work presents the data on mutagenic effects of heavy metal salts (Zn and Cd) on Salmonella typhimurium test strains using mutagenicity test in vitro without metabolic activation and host-mediated assay. The techniques used enabled to determine also the types of mutations arising from the exposure to ZnCl2 and CdCl2.     

Investigation of the mutagenic activity of tobacco smoke

The genotoxic effect of whole tobacco smoke was studied employing the Salmonella/microsome mutagenicity assay, the micronucleus test in mouse bone marrow and UDS in peripheral human lymphocytes. It was established that tobacco smoke (120-480 cm3 in a 16-1 glass chamber, at 1-10 min exposure time) induced a 3-9-fold increase of spontaneous his+ reversion mutation rate in S. typhimurium TA98, but not in strains TA97a, TA100 and TA102. Addition of S9 mix obtained from the liver of Aroclor 1254-treated rats was necessary to reveal the mutagenic activity of tobacco smoke. Treatment of BDF1 mice placed in a 14-1 glass chamber with tobacco smoke (600 cm3 smoke, 2 exposures of 30 min each, with a 1-min interval between them) caused a 2-fold dose-dependent elevation of the number of micronucleated PCE in bone marrow. No cumulative effect was detected when mice were treated with tobacco smoke during 2-28 consecutive days. The effect observed 24 h after tobacco-smoke exposure was abolished 48 h later. Tobacco smoke (180 or 360 cm3) passed through the culture medium (with or without S9 mix) of human peripheral lymphocytes (the cells were then incubated for 60 min at 37 degrees C) did not increase the spontaneous rate of UDS. Both the Salmonella/microsome mutagenicity assay employing S. typhimurium TA98 strain and the micronucleus test in mouse bone marrow might be useful in studying tobacco smoke-induced mutagenesis.     

The mouse bone marrow micronucleus test: evaluation of 21 drug candidates

The mouse bone-marrow micronucleus test is one of the most widely used genetic toxicology assays. In this report the results of testing 21 compounds in the micronucleus test are presented. Of the 21 compounds tested, 3 potential chemotherapeutic agents were identified as strongly clastogenic. In addition, one compound was identified as a weak inducer of micronuclei in the assay. Further testing of this compound in an in vivo bone marrow metaphase analysis failed to confirm this material as clastogenic. The remaining 17 compounds were classified as negative in the assay. In general the results of the micronucleus test agreed with the results of other genetic toxicology assays on this group of compounds.     

The effect of gamma-radiation on smoked fish using short-term mutagenicity assays

The effect of gamma-radiation on the mutagenicity potential of wood-smoked fish (Rastrelliger sp.) was investigated. Smoked fish were irradiated with radiation doses of 2.0, 4.0, 6.0 and 8.0 kGy. The DMSO extracts of non-radiated and irradiated smoked fish were tested for mutagenicity using the Ames plate incorporation assay, host-mediated assay, and the micronucleus test. It was observed that gamma-irradiation did not induce any significant increase in the number of revertants of TA98, TA100 and TA104 as compared with the non-radiated smoked fish. Results of the host-mediated assay and the micronucleus test showed no difference in the mutagenic response of non-radiated and irradiated smoked fish. The results indicate that gamma-radiation does not introduce mutagens in smoked fish.     

The possible role of probiotics as dietary antimutagen.

Possible antimutagenic actions of probiotics--mainly lactic acid bacteria--were examined using in vitro and in vivo test systems. In the Ames test with Salmonella typhimurium TA1538 beef extract and nitrosated beef extract were used as mutagens. L. casei showed high antimutagenic activity on mutagenicity induced by nitrosated beef extract only without S9 mix, whereas Omniflora (a lyophilized preparation of lactobacilli and E. coli) and its cell-free culture broth exhibited antimutagenic action only on beef extract. The actions of probiotics were more homogeneous when living animals were used in the tests. Using busulfan as a mutagen both the chromosome aberration test (with Chinese hamster bone marrow cells) and the micronucleus test (with bone marrow cells of Chinese hamsters and mice) showed strong anticlastogenic action when L. casei, Omniflora or yoghurt (with living bifiobacteria) were given orally at the same time as the mutagen. Lactobacilli were effective also after i.p. injection. Cell-free culture broths had no or only weak antimutagenic effects. Mutagen-induced chromosome aberrations and micronuclei were reduced by up to 80% by the lactobacilli.     

Host-mediated mutagenicity experiments with benzo[a]pyrene and two of its metabolites

Benzo[a]pyrene (BP) and two of its major metabolites, the ultimate mutagen BP-4,5-oxide and the proximate mutagen trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-diol) were investigated for mutagenicity in Salmonella typhimurium TA1538, TA98 and TA100 using an intrasanguineous host-mediated assay. BP and BP-4,5-oxide were not mutagenic under any experimental conditions. BP-7,8-diol was inactive with the strain TA1538 but was mutagenic with the strains TA98 and TA100. The effect was potentiated by pretreatment of the host mice with the cytochrome P-450 inducer 5,6-benzoflavone. We conclude: (i) one of the reasons for the observed insensitivity of the intrasanguineous host-mediated assay towards BP is that BP-4,5-oxide, which contributes to the microsome-mediated mutagenicity of BP, is inactive in the host-mediated assay; (ii) the finding that BP-7,8-diol is mutagenic in the host-mediated assay demonstrates that the lack of mutagenicity of BP is not intrinsic; (iii) the potentiated mutagenicity after treatment of the hosts with 5,6-benzoflavone suggests that cytochrome P-450 is more important in the activation of BP-7,8-diol in this system than other enzymes (e.g. prostaglandin synthase) that can also activate this compound in vitro.     

Practical aspects of mutagenicity testing strategy: an industrial perspective

Genetic toxicology studies play a central role in the development and marketing of new chemicals for pharmaceutical, agricultural, industrial, and consumer use. During the discovery phase of product development, rapid screening tests that require minimal amounts of test materials are used to assist in the design and prioritization of new molecules. At this stage, a modified Salmonella reverse mutation assay and an in vitro micronucleus test with mammalian cell culture are frequently used for screening. Regulatory genetic toxicology studies are conducted with a short list of compounds using protocols that conform to various international guidelines. A set of four assays usually constitutes the minimum test battery that satisfies global requirements. This set includes a bacterial reverse mutation assay, an in vitro cytogenetic test with mammalian cell culture, an in vitro gene mutation assay in mammalian cell cultures, and an in vivo rodent bone marrow micronucleus test. Supplementary studies are conducted in certain instances either as a follow-up to the findings from this initial testing battery and/or to satisfy a regulatory requirement. Currently available genetic toxicology assays have helped the scientific and industrial community over the past several decades in evaluating the mutagenic potential of chemical agents. The emerging field of toxicogenomics has the potential to redefine our ability to study the response of cells to genetic damage and hence our ability to study threshold phenomenon.     

Considerations in the U.S. Environmental Protection Agency's testing approach for mutagenicity.

OPP: This paper provides the rationale and support for the decisions the OPP will make in requiring and reviewing mutagenicity information. The regulatory requirement for mutagenicity testing to support a pesticide registration is found in the 40 CFR Part 158. The guidance as to the specific mutagenicity testing to be performed is found in the OPP's Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals (referred to as the Subdivision F guideline). A revised Subdivision F guideline has been presented that becomes the current guidance for submitters of mutagenicity data to the OPP. The decision to revise the guideline was the result of close examination of the version published in 1982 and the desire to update the guidance based on developments since then and current state-of-the-science. After undergoing Agency and public scrutiny, the revised guideline is to be published in 1991. The revised guideline consists of an initial battery of tests (the Salmonella assay, an in vitro mammalian gene mutation assay and an in vivo cytogenetics assay which may be either a bone marrow assay for chromosomal aberrations or for micronuclei formation) that should provide an adequate initial assessment of the potential mutagenicity of a chemical. Follow-up testing to clarify results from the initial testing may be necessary. After this information as well as all other relevant information is obtained, a weight-of-evidence decision will be made about the possible mutagenicity concern a chemical may present. Testing to pursue qualitative and/or quantitative evidence for assessing heritable risk in relation to human beings will then be considered if a mutagenicity concern exists. This testing may range from tests for evidence of gonadal exposure to dominant lethal testing to quantitative tests such as the specific locus and heritable translocation assays. The mutagenicity assessment will be performed in accordance with the Agency's Mutagenicity Risk Assessment Guidelines. The mutagenicity data would also be used in the weight-of-evidence consideration for the potential carcinogenicity of a chemical in accordance with the Agency's Carcinogen Risk Assessment Guidelines. In instances where there are triggers for carcinogenicity testing, mutagenicity data may be used as one of the triggers after a consideration of available information. It is felt that the revised Subdivision F guideline will provide appropriate, and more specific, guidance concerning the OPP approach to mutagenicity testing for the registration of a pesticide. It also provides a clearer understanding of how the OPP will proceed with its evaluation and decision making concerning the potential heritable effects of a test chemical.(ABSTRACT TRUNCATED AT 400 WORDS)     

The mutagenicity of hydrazine and some of its derivatives.

The mutagenic effect of ethylenethiourea (ETU), a degradation product and metabolite of ethylenebisdithiocarbamates, which are widely used as fungicides, was studied in different test systems.ETU induced mutations of the base-pair substitution type in Salmonella typhimurium TA 1530 in vitro as well as in the host-mediated assay. In the host-mediated assay, a dose of 6000 mg/kg (LD₅₀ = 5400 mg/kg) resulted in a slight but significant increase of the reversion frequency by a factor of 2.37.The results of the micronucleus test were negative after two-fold oral applications of 700, 1850 and 6000 mg/kg to Swiss albino mice. Thus it is concluded that ETU hardly induces any chromosomal anomality in the bone marrow.No dominant-lethal effect was observed after single oral doses of 500, 1000 and 3500 mg/kg given to male mice.     

Absence of mutagenic activity of WHR-1142A, lidamidine hydrochloride, in 4 short-term tests for mutagenic/carcinogenic potential

WHR-1142A, lidamidine hydrochloride, an antidiarrhoeal agent, was tested for possible mutagenic/carcinogenic activity in the Ames Salmonella typhimurium/metabolic activation test, the micronucleus test, by analysis of metaphase chromosomes obtained from human lymphocytes grown in culture and in a cell transformation assay. No evidence of mutagenic/carcinogenic activity due to WHR-1142A, lidamidine hydrochloride, was found in any of the 4 tests.     

Concomitant observations of UDS in the liver and micronuclei in the bone marrow of rats exposed to cyclophosphamide or 2-acetylaminofluorene

Oral dosing of between 5–30 mg/kg of cyclophosphamide (CP) to Alderley Park rats induced micronuclei in the bone marrow between 12 and 36 h after dosing, but failed to induce unscheduled DNA synthesis (UDS) in the liver at similar dose levels and treatment periods. Dose levels of > 30 mg/kg were toxic to the liver. In contrast, 2-acetylaminofluorene (2AAF) induced UDS in the rat liver between 4–36 h after dosing, but gave only a weak response in the bone marrow assay at dose levels between 0.5 and 2 g/kg. Selected observations were made for each chemical using both tissues of the same test animal.

It is concluded that an assessment of the genotoxicity in vivo of chemicals defined as genotoxic in vitro will contribute to an assessment of their possible mammalian carcinogenicity, and that these should involve assays conducted using both the bone marrow and the liver of rodents. Due to its relative ease of commission, the bone marrow micronucleus assay will usually be conducted first; in the case of negative results it is recommended that a liver genotoxicity assay should also be conducted. The case for employing in vivo short-term genotoxicity tests to predict the possible organotropic carcinogenicity or germ cell mutagenicity of a new in vitro genotoxin is discussed.     

Genotoxicity of microcystic cyanobacteria extract of a water source in China.

The water pollution of toxic cyanobacteria (blue-green algae) is a worldwide problem and worsens with industrialization. Microcystins are potent cyclic heptapeptidic hepatotoxins produced mainly by Microcystis aeruginosa, and their hepatotoxicity has been well-documented. In contrast, information on the genotoxic effects of microcystins is relatively scarce. In our present study, the genotoxicity of microcystic cyanobacteria extract (MCE) of a water source in China was studied using Salmonella typhimurium assay (Ames test), comet assay (Single cell gel electrophoresis) and mouse micronucleus test. Results from Ames test indicated that MCE had strong mutagenicity regardless of the presence of S9. Moreover, MCE was able to induce DNA damage in primary cultured rat hepatocytes examined by comet assay. In addition, MCE also enhanced bone marrow micronucleated polychromatic erythrocytes in mice. The analysis of HPLC showed that the main component of MCE was microcystin-LR. The understanding of the potent genotoxicity of MCE will help to establish the possible link between water cyanobacteria contamination and high risk of primary liver cancer found in some endemic areas.     

Mutagenicity study of fried sausages in Salmonella, Drosophila and mammalian cells in vitro and in vivo

The basic extract of pan-fried sausages was studied for mutagenic potential in seven test systems. Mutagenic activity was high in the standard Ames assay in the Salmonella typhimurium strains TA1538 and TA98 in presence of S9 mix. In vivo, in the intrasanguine host-mediated assay with strain TA98 on Aroclor-pretreated mice, the mutagenic activity of the extract was low. A borderline activity was seen in the SCE assay in vitro with V79 Chinese hamster cells in presence of S9 mix. No significant mutagenic action was found in the gene-mutation assay for thioguanine resistance with V79 cells, the Drosophila sex-linked recessive lethal test, the micronucleus test and the mammalian spot test.     

Diminished genotoxicity of mitomycin C and farmorubicin included in polybutylcyanoacrylate nanoparticles.

The genotoxic effects of mitomycin C (MMC) and farmorubicin (FR) in a free form and included in polybutylcyanoacrylate nanoparticles (PBCN) were studied employing the Salmonella/microsome mutagenicity assay and the micronucleus test in mouse bone marrow as well as in mouse fetal liver. The data obtained clearly indicated that MMC (0.25-2.00 micrograms/plate) was a strong mutagen in S. typhimurium TA102, while the same concentrations of this compound in PBCN were ineffective in inducing his+ revertant mutations in bacterial cells. A similar total suppression of mutagenic activity of FR (1.0-20.0 micrograms/plate) was registered in S. typhimurium TA98 when the drug was included in PBCN. Furthermore, the incorporation of MMC (2.0 or 4.0 mg/kg, i.p.) into PBCN strongly diminished or even abolished its clastogenic activity in the bone marrow of virgin and pregnant mice as well as in mouse fetal liver, respectively. In addition, a lack of genotoxic effect of PBCN only was also established. The toxic activity of MMC in mouse bone marrow was significantly reduced or completely abolished after its inclusion in PBCN. A conclusion might be drawn that the genotoxic activity of some antitumor drugs might be markedly diminished or even abolished after their incorporation in PBCN.     

Re-evaluation of the mutagenic potential of quinacrine dihydrochloride dihydrate.

Quinacrine has been used for voluntary female non-surgical sterilization for its ability to produce tubal occlusion. Safety issues regarding quinacrine have been raised because it has been shown to intercalate with DNA. Therefore, safety issues need to be resolved by appropriate toxicology studies to support a review for human transcervical use. Such toxicology studies include mutagenicity assays. Here we report an evaluation of the genotoxicity of quinacrine dihydrochloride dihydrate (QH) using a battery of assays. In the bacterial mutagenicity assay, QH was strongly positive in Salmonella typhimurium tester strain TA1537 with and without S9-activation and in S. typhimurium tester strain TA98 with S9-activation; QH was also strongly positive in Escherichia coli WP2 uvrA without S9-activation. QH was not mutagenic in S. typhimurium tester strains TA100 and TA1535 with and without S9-activation. QH was mutagenic in the mouse lymphoma assay in the absence of S9-activation. QH was clastogenic in Chinese hamster ovary (CHO) cells, with and without S9-activation. QH was negative for polyploidy in the same chromosome aberration test. Using a triple intraperitoneal injection treatment protocol in both male and female mice, QH was negative in the in vivo mouse micronucleated erythrocyte (micronucleus) assay. These results confirm that QH is mutagenic and clastogenic in vitro and suggest a potential risk to human health due to QH exposure after intrauterine exposure.     

Comutagenic and coclastogenic effects of selenium in vitro and in vivo

The comutagenic activity of selenium was investigated using in vitro and in vivo techniques, including the liquid suspension modification of the standard Salmonella/microsome mutagenicity assay, the metaphase analysis of chromosome aberrations in CHO cells and in mouse bone marrow as well as the micronucleus assay in mouse bone marrow. 4 h growth of S. typhimurium TA1535 in a nutrient broth containing 2.9 x 10(-5) M but not 1.16 x 10(-5) M Na2SeO3 caused an up to 10-fold increase of the number of N-methylnitrosourea (MNU, 2.0-2.5 mM)-induced his+ revertants and an up to 2-fold elevation of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 1.48 x 10(-5))-induced mutation rate. Pretreatment of bacteria with Na2SeO3 alone had no effect on the spontaneous mutation level. The combined treatment of CHO cells with MNNG (1.25 x 10(-5) M) or tobacco smoke (TS, 2-3 puffs generated by a cigarette inhalation machine) plus Na2SeO3 (0.58-1.16 x 10(-5) M) starting 2 h and 4 h before the MNNG or TS treatment respectively resulted in a 2-3-fold increase in the percent of metaphases with chromosome aberrations. Furthermore, treatment for 7-14 days of male BDF1 (C57Bl x DBA2) or CC57W mice with Na2SeO3, added to the drinking water at a concentration of 10 ppm, potentiated by 2-3 times the chromosome-damaging activity of urethane (0.5-1.0 g/kg, i.p.) in mouse bone marrow, as measured by the formation of micronuclei or chromosome aberrations. In addition, Na2SeO3 increased up to 43.8% the number of micronucleated polychromatic erythrocytes (MNPCE) induced by mitomycin C (MMC, 1.5 mg/kg, i.p.) in BDF1 mouse bone marrow. Treatment of mice with Na2SeO3 alone had no effect on the spontaneous level of MNPCE. All these findings are consistent with a comutagenic and coclastogenic activity of selenium both in prokaryotes and in eukaryotes, in vitro as well as in vivo after pretreatment of target cells with the trace element.     

Absence of mutagenicity effects of Psidium cattleyanum Sabine (Myrtaceae) extract on peripheral blood and bone marrow cells of mice

Cattley guava (Psidium cattleyanum Sabine) is a native fruit of Brazil that is popular both as a sweet food and for its reputed therapeutic properties. We examined whether it could damage DNA using the alkaline single-cell gel electrophoresis (comet assay) and the micronucleus test in leukocytes and in bone marrow cells of mice. P. cattleyanum leaf extract was tested at concentrations of 1000, 1500 and 2000 mg/kg. N-nitroso-N-ethylurea was used as a positive control. Peripheral blood leukocytes were collected 4 and 24 h after the treatments for the comet assay, and bone marrow cells were collected after 24 and 48 h for the micronucleus test. Unlike N-nitroso-N-ethylurea, P. cattleyanum extract failed to induce a significant increase in cell DNA damage, in micronucleated cell frequency, and in bone marrow toxicity. The lack of mutagenicity and cytotoxicity with high doses of this plant extract means that it can be safely used in traditional medicine.     

Genotoxicity of o-aminoazotoluene (AAT) determined by the Ames test, the in vitro chromosomal aberration test, and the transgenic mouse gene mutation assay

o-Aminoazotoluene (AAT) has been evaluated as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). The Ames test found it to be mutagenic in the presence of a metabolic activation system, whereas it has little clastogenicity either in vitro or in vivo in the chromosomal aberration assay. AAT is also carcinogenic in the lung or liver of mice and rats given long-term administrations. Therefore, metabolites generated in the liver etc. may have gene mutation activity, and carcinogenesis would occur. We examined the mutagenicity of AAT in a gene mutation assay, using lacZ transgenic mice (MutaMice) and a positive selection method. AAT showed positive results for organs with metabolic functions, such as liver and colon and other organs. Positive results were also seen in an Ames test in the presence of metabolic activation and negative results seen in a chromosomal aberration test. Therefore, AAT had the potential to cause gene mutation in the presence of metabolic activation systems in vitro and the same reaction was confirmed in vivo with organs with metabolic function, such as liver and colon, but little clastogenicity in vitro or in vivo. Thus, metabolites with gene mutation activity may be responsible for the carcinogenicity of AAT. The transgenic mouse mutation assay proved to be useful for concurrent assessment of in vivo mutagenicity in multiple organs and to supplement the standard in vivo genotoxicity tests, such as the micronucleus assay which is limited to bone marrow as the only target organ.     

Assessment of the utility of the micronucleus test for petroleum-derived materials

The micronucleus test is a commonly used in vivo assay for chromosomal damage and is an integral part of many mutagenicity testing strategies. The present report describes an assessment of the micronucleus test for the detection of mutagenic potential of petroleum-derived materials. To this end, studies were conducted with catalytically cracked clarified oil (CCCO). This material contains high levels of polycyclic aromatic constituents (PAC) and is a very potent inducer of mouse skin tumors. CCCO is also active in the Salmonella assay and other in vitro tests. As CCCO is the most potent of the various petroleum-derived materials in other assays, it was assumed to be the most easily detectable in the micronucleus test. CCCO was tested in standard mouse micronucleus tests utilizing oral and intraperitoneal injection for test material administration. All of these studies were negative, although DMBA, tested at roughly equivalent levels based on potency in the Salmonella assay, produced statistically significant increases in micronucleus frequency. In a second series of studies, aromatic fractions of CCCO were prepared and tested at up to acutely toxic levels. Results of these studies were also negative. Finally, another petroleum-derived material which is carcinogenic and contained PAC was tested in the micronucleus assay. It also produced negative results. Thus, it was concluded that petroleum-derived materials do not produce clastogenic effects in vivo in the mouse micronucleus test, despite the fact that some pure polycyclic aromatic hydrocarbons are quite active in this assay.