Aloe-emodin, a hydroxyanthracene derivative, is not genotoxic in an in vivo comet test.

Aloe-emodin, one of the molecules belonging to the group of hydroxyanthracene derivatives, was recently described as genotoxic in vivo. Indeed, the EFSA judged that aloe-emodin, together with other similar molecules (emodin and danthron) and extracts from the leaf of Aloe species containing hydroxyanthracene derivatives, could represent a risk factor for colorectal cancer mediated by a genotoxic effect. Given the marked uncertainty regarding the conclusions in the opinion of the EFSA ANS Panel and conflicts in the epidemiological data on which the opinion is based, a new in vivo study (in vivo alkaline comet assay in mice - OECD 489) was conducted to test the potential genotoxicity of aloe-emodin at doses of 250, 500, 1000 and 2000 mg/kg bw/day on preparations of single cells from the kidney and colon of treated male mice. Following treatment with the test item, no clinical signs were observed in animals in any treatment group. Slight body-weight loss was randomly observed in all groups treated with the test item and was more evident in the groups dosed at 1000 and 2000 mg/kg bw/day. Under these experimental conditions, aloe-emodin showed no genotoxic activity. Possible oxidative damage to colon tissues could not be excluded based on the results obtained after repair enzyme treatment.

Oxidative Stress, Mutations and Chromosomal Aberrations Induced by In Vitro and In Vivo Exposure to Furan.

Furan is a volatile compound that is formed in foods during thermal processing. It is classified as a possible human carcinogen by international authorities based on sufficient evidence of carcinogenicity from studies in experimental animals. Although a vast number of studies both in vitro and in vivo have been performed to investigate furan genotoxicity, the results are inconsistent, and its carcinogenic mode of action remains to be clarified. Here, we address the mutagenic and clastogenic activity of furan and its prime reactive metabolite cis-2 butene-1,4-dial (BDA) in mammalian cells in culture and in mouse animal models in a search for DNA lesions responsible of these effects. To this aim, Fanconi anemia-derived human cell lines defective in the repair of DNA inter-strand crosslinks (ICLs) and Ogg1-/- mice defective in the removal of 8-hydroxyguanine from DNA, were used. We show that both furan and BDA present a weak (if any) mutagenic activity but are clear inducers of clastogenic damage. ICLs are strongly indicated as key lesions for chromosomal damage whereas oxidized base lesions are unlikely to play a critical role.

Overview of the marmoset as a model in nonclinical development of pharmaceutical products.

Callithrix jacchus (common marmoset) is one of the more primitive non-human primate species and is used widely in fundamental biology, pharmacology and toxicology studies. Marmosets breed well in captivity with good reproductive efficiencies and their sexual maturity is reached within 18 months of age allowing for rapid expansion of colonies and early availability of sexually mature animals permitting an earlier assessment of product candidates in the adult. Their relatively small size allows a reduction in material requirements leading to a reduction in development time and cost. Fewer animals are also required due to their ability to be used in both pharmacology and toxicology (nonclinical) studies. These factors, alongside a better understanding of their optimal nutrient and welfare requirements over recent years, facilitate the generation of a more cohesive and robust dataset. With the growth of biotechnology-derived pharmaceuticals, non-human primate use has, by necessity, also increased; nevertheless, there is also a growing public call for minimizing their use. Utilizing, the more primitive marmoset species may provide the optimal compromise and once the scientific rationale has been carefully considered and their use justified, there are several advantages to using the marmoset as a model in nonclinical development of pharmaceutical products.

Lack of in vivo genotoxic effect of dried whole Aloe ferox juice.

Aloe ferox Mill is widely used as a traditional herbal medicine for the treatment of a broad spectrum of illnesses given its laxative, anti-inflammatory, bitter tonic, anti-oxidant, antimicrobial and anti-cancer properties. Using the in vivo alkaline comet assay in animals (OECD 489), this study investigated the potential in vivo genotoxicity of dried Aloe ferox juice at dose levels of 500, 1000, and 2000 mg/kg/day in mice. Aloe ferox showed no genotoxic activity in preparations of single cells from the colon of the treated Hsd:ICR (CD-1) male mice. No statistically significant increase in DNA migration over the negative control was observed by analysis of variance for both comet parameters, tail moment and tail intensity, apart from the positive control ethyl methanesulphonate that induced clear and statistically significant increases in DNA migration parameters over the concurrent controls. The new reported scientific evidence unequivocally demonstrates that dried Aloe ferox juice containing hydroxyanthracene derivatives does not induce DNA damage in preparations of single cells from colon in in vivo comet genotoxicity studies. This suggests that the hyperplastic changes and mucosal hyperplasia observed after long-term administration of Aloe vera non-decolourised whole leaf extract may be attributed to an epigenetic effect of the material under investigation.

In Vitro Cytogenetic Assays: Chromosomal Aberrations and Micronucleus Tests.

Chromosome damage is a very important indicator of genetic damage relevant to environmental and clinical studies. Detailed descriptions of the protocols used for detection of chromosomal aberrations induced by genotoxic agents in vitro both in the presence or absence of rat liver-derived metabolizing systems are given in this chapter. Structural chromosomal aberrations that can be observed and quantified at metaphases are described here. For the detection of chromosomal damage (fragments or whole chromosome) in interphase, the micronucleus test can be used, and a description of this test is also presented. Criteria for determining a positive result using appropriate statistical methods are described.

In vitro cytogenetic results supporting a DNA nonreactive mechanism for ochratoxin A, potentially relevant for its carcinogenicity.

Ochratoxin A (OTA) is a widespread mycotoxin of cereals and many agricultural products and causes high incidences of renal tumors in rodents. Although its carcinogenic properties have been known since the eighties, the precise mechanism of action is still relatively undefined. At present, increasing evidence suggests that OTA does not act with a direct genotoxic mechanism, opposed to other previous evidence where the formation of DNA adducts by 32P-postlabeling was observed. The genotoxic activity of OTA assessed in a variety of in vitro and in vivo studies was very low if genotoxic at all. In this study, we clearly show that OTA does not bear any clastogenic or aneugenic activity based on the absence of the induction of chromosome aberrations, sister chromatid exchanges, and micronuclei in human lymphocytes and V79 cells in vitro in both the absence and the presence of S9 metabolism. Alternatively, cytogenetic analyses evidenced significant increases in endoreduplicated cells and highly condensed metaphases with separated chromatids. This implies that OTA or its possible metabolites do not covalently bind DNA through the formation of adducts since structural chromosome aberrations are a very sensitive end points to detect chemical carcinogens with electrophilic substituents. Alternatively, induction of endoreduplication and chromatid separation provides strong evidence for a DNA nonreactive mechanism of OTA carcinogenicity involving the disruption of mitosis by interfering with key regulators of chromosome separation and progression of mitosis. This causes a temporary arrest of mitoses and premature exit from it (mitotic slippage) to generate endoreduplication and polyploidy accompanied by increased risk of aneuploidy and subsequent tumor formation.

SFTG international collaborative study on in vitro micronucleus test II. Using human lymphocytes.

This study on the in vitro micronucleus assay, comprising 11 laboratories using human lymphocytes, was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Nine coded substances were assessed for their ability to induce micronuclei in human lymphocytes in vitro, mitomycin C being used as a positive control. Cultures were exposed to the test substances for a short (early or late) time or for a long time, followed by a short or long recovery period, in the presence of cytochalasin B. Each chemical was evaluated, generally in two laboratories, using three treatment schedules at least twice. The data were assessed for acceptability, and then classified as negative, positive or equivocal. Two of seven genotoxic compounds, namely colchicine and bleomycin, clearly induced micronuclei. Reproducible results were difficult to obtain for some substances, which tended to be those acting at specific stages of the cell cycle. Cytosine arabinoside, diethylstilboestrol and 5-fluorouracil were classified as equivocal. Urethane and thiabendazole were classified as negative. The two presumed non-genotoxic compounds, mannitol and clofibrate, did not induce micronuclei. Repeat testing, exposing cells at both an early and late time after mitogenic stimulation, was needed to detect substances classified as equivocal. These results show the importance of achieving sufficient inhibition of nuclear division to avoid the possibility of missing an effect. The evaluation of micronuclei in mononucleated as well as binucleated cells was particularly useful to detect aneugens. There were no false positive results using lymphocytes, indicating a high specificity. It is concluded that the clastogenic or aneugenic potential in vitro of the substances tested was correctly identified in this study, but that refining the protocol to take into account factors such as the stages of the cell cycle exposed to the compound, or the duration of recovery would be likely to improve the sensitivity of detection using lymphocytes.

Use of chromosome painting for detecting stable chromosome aberrations induced by melphalan in mice.

Chromosomal aberrations are a measure of genomic instability, which is known to play a key role in the initiation and promotion of carcinogenesis. Stable reciprocal translocations are of particular importance since they are often involved in neoplastic transformation and tumor cell clonal evolution. In this study, chromosome painting analysis was used to test for stable aberrations induced in the bone marrow of C57BL/6J and FVB mice exposed for 4 weeks to 2 or 4 mg/kg of melphalan (MLP), a chemotherapeutic agent with carcinogenic potential. To compare the chemical-induced damage in different tissues, chromosome aberrations were also analyzed by chromosome painting in the spleen of C57BL/6J mice. At the 2 mg/kg dose, MLP induced comparable levels of chromosome-type aberrations in bone marrow cells of both mouse strains and in splenocytes of C57BL/6J mice. At 4 mg/kg, no further increase in aberrations was detected in bone marrow, while a dose-effect relationship was found in spleen cells. This different response may result from a negative selection against highly damaged bone marrow cells during mitotic proliferation. The results indicate that chromosome painting is a useful tool for detecting stable chromosome aberrations in somatic cells exposed to MLP and possibly to other genotoxic chemical carcinogens.

Melphalan-induced DNA damage in p53(+/-) and wild type mice analysed by the comet assay.

Melphalan is an alkylating substance used as a therapeutic agent; its mutagenicity is related to its ability to produce monoadducts and to form DNA cross-links. The alkaline comet assay is a useful test for the detection of DNA lesions. However, cross-links are not easily detected under standard conditions. Recently, modifications to the test have been introduced to measure cross-links by evaluating the reduction in induced DNA migration. In this work, the standard comet assay and an assay modified by prolonging the electrophoresis time have been applied to evaluate DNA lesions induced by single, 4 or 26 weekly oral administrations of melphalan to p53(+/-) knockout and to isotype parental mice. Cells were analysed from the liver, bone marrow, peripheral blood and the distal intestine. Moreover, a further protocol in which the presence of cross-links was inferred by the reduction in X-ray-induced DNA migration was applied to bone marrow cells and the sensitivity of the different methods was compared. The majority of groups examined by the standard protocol showed no difference compared to controls, while the modified protocol (prolonged electrophoresis time) could detect a retarded DNA migration in cells from all the organs analysed with the exception of bone marrow cells. Only the protocol based on X-ray in vitro irradiation showed the presence of melphalan-induced cross-links in bone marrow cells exposed to 2mg/kg for 4 weeks, demonstrating that this was the most sensitive approach for detecting this type of lesion. DNA lesions were evident in all the organs analysed. However, results suggest that the kinetics of cross-link repair could be different in bone marrow cells compared to other organs tested. After comparison between genotype-matched treated and control groups, a significant effect was shown more frequently in p53(+/-) than in wild type groups.

In vitro cytogenetic assays: chromosomal aberrations and micronucleus tests.

Chromosome damage is a very important indicator of genetic damage relevant to environmental and clinical studies. Detailed descriptions of the protocols used for detection of chromosomal aberrations induced by unknown agents in vitro both in the presence or the absence of rat liver-derived metabolizing systems are given. Structural chromosomal aberrations that can be observed and quantified at metaphases are described here. For the detection of chromosomal damage (fragments or whole chromosome) in interphase, the micronucleus test can be used and a description of this test is also presented. Criteria for determining a positive result using appropriate statistical methods are described.

The local lymph node assay (LLNA).

The murine local lymph node assay (LLNA) is a widely accepted method for assessing the skin sensitization potential of chemicals. Compared with other in vivo methods in guinea pig, the LLNA offers important advantages with respect to animal welfare, including a requirement for reduced animal numbers as well as reduced pain and trauma. In addition to hazard identification, the LLNA is used for determining the relative skin sensitizing potency of contact allergens as a pivotal contribution to the risk assessment process. The LLNA is the only in vivo method that has been subjected to a formal validation process. The original LLNA protocol is based on measurement of the proliferative activity of draining lymph node cells (LNC), as determined by incorporation of radiolabeled thymidine. Several variants to the original LLNA have been developed to eliminate the use of radioactive materials. One such alternative is considered here: the LLNA:BrdU-ELISA method, which uses 5-bromo-2-deoxyuridine (BrdU) in place of radiolabeled thymidine to measure LNC proliferation in draining nodes.

Chromosome aberrations and telomere length modulation in bone marrow and spleen cells of melphalan-treated p53+/- mice.

The p53 gene regulates cell cycle and apoptotic pathways after induction of DNA damage. Telomeres, capping chromosome ends, are involved in maintaining chromosome stability; alterations of their length have been related to increased levels of chromosomal aberrations. To study a possible interaction between chromosome aberrations, telomere dysfunction, and p53, we investigated via painting analysis the induction and persistence of chromosome aberrations in bone marrow and spleen cells of p53+/- (and wild type) mice exposed for 4, 13, or 26 weeks to 2 mg/kg melphalan (MLP), a chemotherapeutic agent with carcinogenic potential. In addition, telomere length was evaluated in bone marrow cells by quantitative fluorescence in situ hybridization (Q-FISH). Chromosome aberrations were significantly increased in both tissues after MLP treatment. The p53 genotype did not influence the response of spleen cells, whereas a slight but significant increase of the aberration frequency was measured in the bone marrow of p53+/- mice exposed to MLP for 13 weeks with respect to the level detected in the matched wild-type group. The main finding of our still preliminary results on telomere length modulation was again a difference between the two genotypes. In bone marrow cells of wild-type mice, MLP treatment was associated with telomere shortening, while in p53+/- mice telomere elongation was the prevalent response to MLP exposure. In agreement with previous literature data, our in vivo study suggests that even the lack of a single functional copy of the p53 gene might have an impact on the quantity and quality of chromosome alterations induced in cycling cells by a clastogenic exposure.

Ochratoxin A-induced mutagenesis in mammalian cells is consistent with the production of oxidative stress.

Ochratoxin A (OTA) is a widespread mycotoxin in food and a powerful nephrocarcinogen in rats. The mutagenicity of OTA has been extensively investigated but with conflicting results, thus leaving open the mechanistic question for OTA carcinogenicity. Here, we examined the mutagenicity of OTA by using well-standardized mutation assays such as the hypoxanthine-guanine phosphoribosyltransferase (HPRT) assay in Chinese hamster V79 cells and the thymidine kinase assay in mouse lymphoma LY5178 cells. OTA-induced HPRT mutations were characterized at the molecular level. In V79 cells, OTA produced a dose- and time-related decrease in cell number as a consequence of the transitory cytostatic effect mediated by G2/M cell cycle arrest. In both mutation assays, OTA was weakly mutagenic and this effect was independent of biotransformation. OTA-induced mutations were characterized by point mutations (48%) and a lack of a detectable reverse-transcription polymerase chain reaction product (52%). The pattern of OTA-induced point mutations was similar to that of spontaneous mutants, suggesting that OTA induced an increase of the endogenous oxidative metabolism but not covalent DNA adducts. Our data support a model where OTA is mutagenic via oxidative DNA damage induction.