Genotoxicity assessment of ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN)

Ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN) are relatively insensitive explosive compounds that are being explored as safe alternatives to other more sensitive compounds. When used in combination with other high explosives they are an improvement and may provide additional safety during storage and use. The genetic toxicity of these compounds was evaluated to predict the potential adverse human health effects from exposure by using a standard genetic toxicity test battery which included: a gene mutation test in bacteria (Ames), an in vitro Chinese Hamster Ovary (CHO) cell chromosome aberration test and an in vivo mouse micronucleus test. The results of the Ames test showed that EDDN increased the mean number of revertants per plate with strain TA100, without activation, at 5000μg/plate compared to the solvent control, which indicated a positive result. No positive results were observed with the other tester strains with or without activation in Salmonella typhimurium strains TA98, TA1535, TA1537, and Escherichia coli strain WP2 uvrA. DETN was negative for all Salmonella tester strains and E. coli up to 5000μg/plate both with and without metabolic activation. The CHO cell chromosome aberration assay was performed using EDDN and DETN at concentrations up to 5000μg/mL. The results indicate that these compounds did not induce structural chromosomal aberrations at all tested concentrations in CHO cells, with or without metabolic activation. EDDN and DETN, when tested in vivo in the CD-1 mouse at doses up to 2000mg/kg, did not induce any significant increase in the number of micronuclei in bone marrow erythrocytes. These studies demonstrate that EDDN is mutagenic in one strain of Salmonella (TA100) but was negative in other strains, for in vitro induction of chromosomal aberrations in CHO cells, and for micronuclei in the in vivo mouse micronucleus assay. DETN was not genotoxic in all in vitro and in vivo tests. These results show the in vitro and in vivo genotoxicity potential of these chemicals.     

In vitro genotoxic evaluation of conventional bleached and biobleached softwood pulp mill effluents

The effluents of pulp and paper mills contain about 300 different chemical compounds; many of them are mutagens and clastogens. Genotoxic studies have shown that chlorination stage liquors are significantly more genotoxic, in the Ames Salmonella assay, than the other process of lignin extraction, and that lyophilized effluents are genotoxic in cultured mammalian cells. Since these effluents from conventional bleaching stages are genotoxic, Chilean industries are interested in changing this process to a less toxic one, such as biobleaching using enzymes. In this study, we tested the in vitro genotoxicity of two types of effluents: an effluent obtained from a conventional radiata pine kraft-bleaching process (effluent D) and one derived from a biobleaching process with hemicellulase (effluent B). Both effluents were tested without any concentration or purification steps in the Ames Salmonella assay (TA100) and in the micronuclei (MN) and sister chromatid exchange (SCE) tests in CHO cells. The results showed that neither effluent induced base pair substitution mutations in the Ames Salmonella assay, and neither increased the micronucleus frequency in CHO cells. But, both increased the SCE frequencies in CHO cells, showing that this assay is more sensitive than the other ones, and that the two effluents contained chemical compounds in amounts enough to induce in vitro genotoxicity measured by the SCE induction.     

The alkaline single cell gel electrophoresis assay with mouse multiple organs: results with 30 aromatic amines evaluated by the IARC and U.S. NTP

The genotoxicity of 30 aromatic amines selected from IARC (International Agency for Research on Cancer) groups 1, 2A, 2B and 3 and from the U.S. NTP (National Toxicology Program) carcinogenicity database were evaluated using the alkaline single cell gel electrophoresis (SCG) (Comet) assay in mouse organs. We treated groups of four mice once orally at the maximum tolerated dose (MTD) and sampled stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow 3, 8 and 24 h after treatment. For the 20 aromatic amines that are rodent carcinogens, the assay was positive in at least one organ, suggesting a high predictive ability for the assay. For most of the SCG-positive aromatic amines, the organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Organ-specific genotoxicity, therefore, is necessary but not sufficient for the prediction of organ-specific carcinogenicity. For the 10 non-carcinogenic aromatic amines (eight were Ames test-positive and two were Ames test-negative), the assay was negative in all organs studied. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative non-genotoxic (Ames test-negative) carcinogens. The alkaline SCG assay, which detects DNA lesions, is not suitable for identifying non-genotoxic carcinogens. The present SCG study revealed a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic non-carcinogens. These results suggest that the alkaline SCG assay can be usefully used to evaluate the in vivo genotoxicity of chemicals in multiple organs, providing for a good assessment of potential carcinogenicity.     

Evaluation of the in vitro genetic toxicity of 4-(2, 4-dichlorophenoxy)butyric acid

The herbicide 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB) is principally used in the USA on peanuts, soybeans and alfalfa. In Europe, it is used on undersown spring barley and grassland (with clover). The genetic toxicity in vitro of the dimethylamine salt of 2,4-DB was examined by employing a range of end points including gene mutation in bacteria (Ames test) and mammalian cell cultures (CHO/HGPRT assay), cytogenetic abnormalities in mammalian cells (CHO/chromosomal aberration assay), and induction of DNA damage and repair in rat hepatocytes. There were no indications of genotoxic potential for 2,4-DB in the first three of these assays. One of the two criteria for a positive response in the UDS assay was exceeded but the increases did not exceed the second criteria for a positive response. The test material was therefore evaluated as weakly active in this assay. The weight of the evidence clearly indicates that 2, 4-DB is not genotoxic to mammals and are consistent with the reported lack of carcinogenic potential for 2,4-DB in both mice and rats.     

Evaluation of a genotoxicity test measuring DNA-strand breaks in mouse lymphoma cells by alkaline unwinding and hydroxyapatite elution

A rapid genotoxicity test, based on the measurement of the proportion of single- to double-stranded DNA by alkaline unwinding and hydroxyapatite elution in mouse lymphoma cells treated in vitro with various chemicals, was evaluated. Seventy-eight compounds from diverse chemical groups, including commonly tested mutagens, toxic compounds not usually tested for genotoxicity and non-toxic compounds not thought to be genotoxic were tested. The results obtained were compared with those from the mouse lymphoma TK locus forward-mutation assay, providing a basis for assessing the relative sensitivity of the 2 assays using the same cells exposed to chemicals under similar conditions. Clear evidence of DNA-damaging activity was obtained with 43 of the compounds, while 4 gave equivocal results. Of the remaining 31 compounds, 14 were toxic without inducing DNA damage while the rest were non-toxic and did not induce any DNA damage. Results were available from both the alkaline unwinding assay and the mouse lymphoma assay for 61 compounds; they showed a concordance between the 2 assays of 77%. Of the 47 compounds that were positive or equivocal in the alkaline unwinding assay, only carbon tetrachloride and prednisolone were negative in the mouse lymphoma assay, while 12 of the 19 compounds that were negative in the alkaline unwinding assay were positive in the mouse lymphoma assay. These included 3 compounds that interfere with nucleic acid metabolism, and 3 crosslinking agents, which would be expected to produce mutations to a greater extent than strand breaks. The other 6 compounds were anthranilic acid, benzoquinone, p-chloroaniline, diethylmaleate, glucose and procarbazine HCl. Of these only the last is a known carcinogen. It is concluded from the present study that there was good overall agreement between the results of the DNA alkaline unwinding and mouse lymphoma TK locus assays, but that the sensitivity of the alkaline unwinding assay is lower for some classes of compounds. Bearing this in mind, the alkaline unwinding assay is considered suitable as a rapid screen for genotoxic activity in eukaryotic cells.     

In vitro genotoxicity of para-phenylenediamine and its N-monoacetyl or N,N'-diacetyl metabolites

para-Phenylenediamine (PPD), a widely used ingredient of oxidative hair dyes, is converted by human hepatocytes and in the human epidermis, or after topical application to rats, to its N-monoacetylated (MAPPD) and/or N,N'-diacetylated (DAPPD) derivatives. We investigated in vitro genotoxic properties of PPD, MAPPD and DAPPD in the Ames test, the micronucleus test (MNT) in human lymphocytes and the mouse lymphoma assay (Hprt locus, PPD only). Given that MAPPD and DAPPD are actual human skin and hepatic metabolites of PPD and represent the substances to which humans are systemically exposed, they were tested in the absence of metabolic activation. In the Ames test, PPD was slightly mutagenic in Salmonella typhimurium strain TA98 in the presence of a rat liver metabolic activation system (S-9), whereas MAPPD and DAPPD were negative in all strains. When tested up to toxic doses, PPD did not induce mutation at the Hprt locus of L5178Y mouse lymphoma cells in two independent experiments, either in the absence or presence of S-9, suggesting that PPD is non-mutagenic in mammalian cells. In the in vitro micronucleus test, PPD induced micronuclei (MN) in cultured human peripheral blood lymphocytes (HL) in the presence of S-9, when tested following 24-h PHA stimulation. No increases in MN frequency were observed in the absence of S-9, when tested following 24-h PHA stimulation. However, PPD induced MN both in the absence and presence of metabolic activation, when tested following 48-h PHA stimulation. In contrast, MAPPD and DAPPD did not induce MN in HL when tested up to 10mM concentrations or to their limit of solubility, respectively, after either 24- or 48-h stimulation. In conclusion, the results of the Ames and MN tests confirm that PPD has a slight genotoxic potential in vitro, although it was non-mutagenic in mammalian cells. Given that MAPPD and DAPPD were negative in the Ames and the MN tests, these acetylated conversion products are considered to be detoxified metabolites that are biologically less reactive than the parent molecule PPD.     

The aromatic amine carcinogens o-toluidine and o-anisidine induce free radicals and intrachromosomal recombination in Saccharomyces cerevisiae

Aniline-based aromatic amine carcinogens are poorly detected in short-term mutagenicity assays such as the Salmonella reverse mutation (Ames) assay. More information on the mechanism of toxicity of such Salmonella-negative carcinogens is needed. Aniline and o-toluidine are negative in the Ames assay, but induce deletions (DEL) due to intrachromosomal recombination in Saccharomyces cerevisiae with an apparent threshold. We show here that the DEL assay also detects the genotoxic activity of another aromatic amine carcinogen, o-anisidine, which is also negative in the Salmonella assay. We also show that the DEL assay distinguishes between o-anisidine and its non-carcinogenic structural analog 2, 4-dimethoxyaniline. We have investigated whether the ability of the DEL assay to detect the carcinogens and to distinguish between the carcinogen/non-carcinogen pair is linked to rises in intracellular free radical species following exposure to the carcinogens. Toxicity induced by all three compounds was reduced in the presence of the free radical scavenger and antioxidant N-acetyl cysteine, recombination induced by o-anisidine and o-toluidine was also reduced by N-acetyl cysteine. All three compounds induced oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate. Superoxide dismutase-deficient strains, however, were hypersensitive to cytotoxicity induced by o-toluidine and o-anisidine but not by the non-carcinogen 2,4-dimethoxyaniline, indicating a different potential for generating superoxide radical between the carcinogens and the non-carcinogen analog. The results indicate that the yeast DEL assay is a useful tool for investigating the genotoxic activity of aromatic amine carcinogens.     

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay. Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 microg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 microg/ml for 3 and 20 h in the absence of S9, and for 3h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 microg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 microg/kg, the last dose being administered 2h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays. TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.     

Genotoxicity evaluation of sesamin and episesamin

Sesamin is a major lignan that is present in sesame seeds and oil. Sesamin is partially converted to its stereoisomer, episesamin, during the refining process of non-roasted sesame seed oil. We evaluated the genotoxicity of these substances through the following tests: a bacterial reverse mutation assay (Ames test), a chromosomal aberration test in cultured Chinese hamster lung cells (CHL/IU), a bone marrow micronucleus (MN) test in Crlj:CD1 (ICR) mice, and a comet assay using the liver of Sprague-Dawley (SD) rats. Episesamin showed negative results in the Ames test with and without S9 mix, in the in vitro chromosomal aberration test with and without S9 mix, and in the in vivo comet assay. Sesamin showed negative results in the Ames test with and without S9 mix. In the in vitro chromosomal aberration test, sesamin did not induce chromosomal aberrations in the absence of S9 mix, but induced structural abnormalities at cytotoxic concentrations in the presence of S9 mix. Oral administration of sesamin at doses up to 2.0g/kg did not cause a significant increase in either the percentage of micronucleated polychromatic erythrocytes in the in vivo bone marrow MN test or in the % DNA in the comet tails in the in vivo comet assay of liver cells. These findings indicate that sesamin does not damage DNA in vivo and that sesamin and episesamin have no genotoxic activity.     

No clastogenic activity of a senna extract in the mouse micronucleus assay.

In previous studies, an analytically well-defined senna extract, commonly used as a laxative, gave positive responses in vitro in the Ames test and in the CHO assay. Therefore, the objective of this study was to investigate the genotoxic activity of the same senna extract in an in vivo genotoxicity assay by means of the generally acknowledged MNT. After administration of an oral dose of 2000 mg senna extract/kg to NMRI mice of both genders, which is equivalent to 119 mg potential rhein/kg, 5.74 mg potential aloeemodin/kg and 0. 28 mg potential emodin/kg, there were no elevated levels of micronuclei in bone marrow cells. Kinetic studies were performed in parallel to demonstrate target organ availability. Highest concentrations in the plasma were reached after 1 h with 3.4 microg rhein/ml and 0.065 microg aloeemodin/ml. In all cases, emodin was below the limit of quantification. From the results, the in vitro clastogenic activity of the senna extract could not be confirmed in the mouse micronucleus assay. Together with further negative in vivo genotoxicity studies with anthranoids, the conclusion can be drawn that there is no indication so far demonstrating a genotoxic risk for patients taking senna laxatives.     

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.     

In vivo Comet assay on isolated kidney cells to distinguish genotoxic carcinogens from epigenetic carcinogens or cytotoxic compounds

The objective of this study was to determine the ability of the alkaline in vivo Comet assay (pH>13) to distinguish genotoxic carcinogens from epigenetic carcinogens when performed on freshly isolated kidney cells and to determine the possible interference of cytotoxicity by assessing DNA damage induced by renal genotoxic, epigenetic or toxic compounds after enzymatic isolation of kidney cells from OFA Sprague-Dawley male rats. The ability of the Comet assay to distinguish (1) genotoxicity versus cytotoxicity and (2) genotoxic versus non-genotoxic (epigenetic) carcinogens, was thus investigated by studying five known genotoxic renal carcinogens acting through diverse mechanisms of action, i.e. streptozotocin, aristolochic acids, 2-nitroanisole, potassium bromate and cisplatin, two rodent renal epigenetic carcinogens: d-limonene and ciclosporine and two nephrotoxic compounds: streptomycin and indomethacin. Animals were treated once with the test compound by the appropriate route of administration and genotoxic effects were measured at the two sampling times of 3-6 and 22-26h after treatment. Regarding the tissue processing, the limited background level of DNA migration observed in the negative control groups throughout all experiments demonstrated that the enzymatic isolation method implemented in the current study is appropriate. On the other hand, streptozotocin, 20mg/kg, used as positive reference control concurrently to each assay, caused a clear increase in the mean Olive Tail Moment median value, which allows validating the current methodology. Under these experimental conditions, the in vivo rodent Comet assay demonstrated good sensitivity and good specificity: all the five renal genotoxic carcinogens were clearly detected in at least one expression period either directly or indirectly, as in the case of cisplatin: for this cross-linking agent, the significant decrease in DNA migration observed under standard electrophoresis conditions was clearly amplified when the duration of electrophoresis was increased up to 40min. In contrast, epigenetic and nephrotoxic compounds failed to induce any signifcant increase in DNA migration. In conclusion, the in vivo rodent Comet assay performed on isolated kidney cells could be used as a tool to investigate the genotoxic potential of a test compound if neoplasic/preneoplasic changes occur after subchronic or chronic treatments, in order to determine the role of genotoxicity in tumor induction. Moreover, the epigenetic carcinogens and cytotoxic compounds displayed clearly negative responses in this study. These results allow excluding a DNA direct-acting mechanism of action and can thus suggest that a threshold exists. Therefore, the current in vivo rodent Comet assay could contribute to elucidate an epigenetic mechanism and thus, to undertake a risk assessment associated with human use, depending on the exposure level.     

Comparative genotoxic effects of the cooked-food-related mutagens Trp-P-2 and IQ in bacteria and cultured mammalian cells

As part of a major study to evaluate the mutagenicity of chemicals produced during the cooking of foods, we examined the responses of bacteria and cultured Chinese hamster cells to the compounds Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) and IQ (2-amino-3-methylimidazo[4,5-f]quinoline), constituents identified in cooked beef and fish. In the Ames/Salmonella tester strain TA1538, both compounds were confirmed to be extremely potent mutagens that were active at levels below 1 ng/plate in the presence of hamster-liver S9 microsomal fraction. 50-fold higher doses of both compounds were required for mutagenicity in the uvr+ tester strain TA1978. Trp-P-2 also behaved as a strong mutagen in CHO cells using the standard exogenous activation with hamster-liver S9 fraction. At concentrations below 1 microgram/ml it produced dose-dependent increases in cell killing, mutations at the hprt and aprt loci, sister-chromatid exchanges, and chromosomal aberrations. An excision-repair-deficient strain was about 2-fold more sensitive than the normal CHO cells with respect to these genotoxic effects of Trp-P-2. IQ had unexpectedly weak activity for all genetic endpoints in the CHO cells, and it produced clear-cut responses only in the repair-deficient cells and only above a concentration of 10 micrograms/ml. The toxicity that was observed with IQ was not affected by the repair capacity of the cells and was not associated with chromosomal aberrations, indicating that damage to cellular structures other than nuclear DNA was likely the predominant pathway for cell killing. Because the culture conditions normally used for CHO cell exposure were shown to be competent in producing bacterial mutagenicity with IQ, it was concluded that the active metabolite of IQ was present in the medium but was somehow ineffective in reaching the DNA of CHO cells and/or reacting with it. These results suggest that the relative mutagenic potency of compounds in Salmonella may bear no direct relationship to relative mutagenicity in CHO cells, emphasizing precaution in attempting to extrapolate microbial data to mammalian somatic cells. This study illustrates the use and merits of a multi-endpoint assay for genetic damage in CHO cells, the utility of using CHO cells that are defective in excision repair of DNA, and the importance of comparative testing between bacterial and mammalian systems.     

Evaluation of the genotoxicity of the imidazole antifungal climbazole: comparison to published results for other azole compounds

Climbazole is an imidazole antifungal agent that can provide anti-dandruff benefits when incorporated into a shampoo matrix. A series of genotoxicity studies were performed to support the human safety of this azole antifungal drug. Climbazole was not mutagenic in the Salmonella typhimurium or Escherichia coli Ames assay and did not induce micronuclei in human lymphocytes. In the mouse lymphoma assay (MLA), climbazole was negative (non-mutagenic) with and without metabolic (S9) activation after a 4 h exposure; an increase in small colony mutants was observed without metabolic activation after a 24 h exposure at concentrations of 15 and 17.5 μg/mL. An in vivo mouse micronucleus test was negative up to a maximum tolerated dose (MTD) of 150 mg/kg climbazole administered orally. In the in vivo/in vitro unscheduled DNA synthesis assay, climbazole showed no evidence of DNA damage in the livers of rats at doses up to the MTD of 200 mg/kg orally. A toxicokinetic study was performed in mice with oral administration of [14C]-climbazole (150 mg/kg). Radioactivity (20.42 μg-equiv./g plasma) was detected 15 min after oral administration of [14C]-climbazole, and the peak concentration was 62.96 μg-equiv./g plasma at 8 h after dosing. The measured amounts of radioactivity in plasma, at all sample times from 15 min up to 24 h, exceeded the concentrations that induced increases in mutation frequency after 24 h exposure of mouse lymphoma cells in vitro (15 and 17.5 μg/mL). These observations lend support to the conclusion that climbazole does not present a genotoxic risk in vivo. Furthermore, these data are consistent with the published data for other azole antifungals that work by preventing the synthesis of ergosterol and, as a class, are generally non-genotoxic, except some isolated positive results of questionable significance. Collectively, these data are supportive of the view that climbazole does not present a genotoxic or carcinogenic risk to humans.     

Genotoxicity of gliotoxin, a secondary metabolite of Aspergillus fumigatus, in a battery of short-term test systems

The genotoxic effects of gliotoxin, a known fungal secondary metabolite, were studied. Gliotoxin was purified from cultivation medium of Aspergillus fumigatus isolated from the indoor air of a moisture problem house. The genotoxicity of gliotoxin was assessed both in bacterial test systems including bacterial repair assay, Ames Salmonella assay and SOS-chromotest, and in mammalian cells using single cell gel (SCG) electrophoresis assay and sister-chromatid exchange (SCE) test. Gliotoxin was found to be genotoxic in the bacterial repair assay but, not in the Salmonella test or SOS-chromotest. A dose-related increase in DNA damage was observed in mouse RAW264.7 macrophages exposed to gliotoxin for 2 h in plain medium in the SCG assay. In contrast to the positive response in the SCG assay, gliotoxin did not induce any clear, dose-related increase in SCEs in Chinese hamster ovary (CHO) cells.     

Niacin, poly(ADP-ribose) polymerase-1 and genomic stability

This study demonstrates that cupric 8-quinolinoxide (CuQ) has induced genetic toxicity in bacteria and mammalian cells through a mechanism of reactive oxygen species (ROS) generation. In the Ames test with rat liver S9, CuQ dose-dependently caused a point mutation in Salmonella typhimurium TA100. The effect of CuQ on DNA damage in HL60 and V79 cells identified in the comet assay is direct and enhanced by the addition of S9. Meanwhile, the tailing length of comet DNA is related to the increasing dosage of CuQ. The genotoxic effect of CuQ on either gene mutation in bacteria or DNA damage in culture cells can be generally blocked by several antioxidants, e.g. pyrrolidinedithiocarbamate, N-acetylcysteine, Vitamins C and E. Supportive of this observation, ROS generation induced by CuQ can be demonstrated both in vitro and in vivo by using the DCFH-DA fluoroprobe. The CuQ-induced intracellular ROS level is also dramatically inhibited by the above antioxidants. Above results imply that the CuQ-induced genotoxicity could be mediated by ROS generation. The nature of ferrous-dependent and S9-enhancing in CuQ-induced ROS generation hints a Fenton-like reaction or some specific enzymes activation could be involved in this process. Furthermore, a DNA damage- and oxidative stress-dependent protein, P53, could also been induced by CuQ treatments in a time-course and dose-dependent manners. Its expression level is recoverable by antioxidants too. In conclusion, our current study strongly suggests that CuQ induces gene mutation, global DNA damage, and P53 expression through a ROS-dependent mechanism.     

Genotoxicity of aloeemodin in vitro and in vivo

The present in vitro and in vivo experiments were undertaken to clarify the genotoxic potential of the hydroxyanthrachinone aloeemodin which can be found in different plant derived products for therapy of constipation. The results demonstrate that aloeemodin is able to induce mutagenic effects in vitro. Positive results were obtained in the chromosomal aberration assay with CHO cells, as well as in the Salmonella reverse mutation assay (frameshift mutations in strains TA 1537, TA 1538 and TA 98). No mutagenic potential of aloeemodin, however, was observed in the gene mutation assay with mammalian cells in vitro (HPRT assay in V79 cells). Each assay was performed in the presence and absence of an extrinsic metabolic activation system (S9-mix). In in vivo studies (micronucleus assay in bone marrow cells of NMRI mice; chromosome aberration assay in bone marrow cells of Wistar rats; mouse spot test [DBA/2J × NMRI]) no indication of a mutagenic activity of aloeemodin was found. Information about a possible reaction of aloeemodin with DNA was derived from an in vivo UDS assay. Hepatocytes of aloeemodin-treated male Wistar rats did not show DNA damage via repair synthesis. All these data suggest that aloeemodin is able to interact with DNA under certain in vitro conditions. However, in vivo the results that were negative did not indicate a genotoxic potential. Therefore, it may be assumed that a genotoxic risk for man might be unlikely.     

Genotoxicity of theobromine in a series of short-term assays

Theobromine (3,7-dimethylxanthine) was evaluated for genotoxic activity in a series of in vitro assays. Theobromine was not mutagenic in the Ames assay up to a maximum concentration of 5000 micrograms/plate either with or without S9 activation. The compound also failed to induce significant levels of chromosome aberrations in CHO cells (with and without S9 activation) or transformation in Balb/c-3T3 cells. At the maximum tolerated concentration theobromine increased the frequency of TK-/- mutants in mouse lymphoma L5178Y cells. Increased frequencies were observed both with and without S9 activation and they were reproducible in 2 independent experiments. Statistically significant increases in SCEs were obtained in human lymphocytes and in CHO cells under nonactivation test conditions. The spectrum of results in this battery of tests indicate that theobromine treatment results in the expression of genotoxic potential in some assays and the observed activity appears qualitatively and quantitatively similar to that of caffeine, a closely related methylxanthine.     

Genotoxicity of the cancer chemopreventive drug candidates CP-31398, SHetA2, and phospho-ibuprofen

The genotoxic activities of three cancer chemopreventive drug candidates, CP-31398 (a cell permeable styrylquinazoline p53 modulator), SHetA2 (a flexible heteroarotinoid), and phospho-ibuprofen (PI, a derivative of ibuprofen) were tested. None of the compounds were mutagenic in the Salmonella/Escherichia coli/microsome plate incorporation test. CP-31398 and SHetA2 did not induce chromosomal aberrations (CA) in Chinese hamster ovary (CHO) cells, either in the presence or absence of rat hepatic S9 (S9). PI induced CA in CHO cells, but only in the presence of S9. PI, its parent compound ibuprofen, and its moiety diethoxyphosphoryloxybutyl alcohol (DEPBA) were tested for CA and micronuclei (MN) in CHO cells in the presence of S9. PI induced CA as well as MN, both kinetochore-positive (Kin+) and -negative (Kin-), in the presence of S9 at ≤100μg/ml. Ibuprofen was negative for CA, positive for MN with Kin+ at 250μg/ml, and positive for MN with Kin- at 125 and 250μg/ml. DEPBA induced neither CA nor MN at ≤5000μg/ml. The induction of chromosomal damage in PI-treated CHO cells in the presence of S9 may be due to its metabolites. None of the compounds were genotoxic, in the presence or absence of S9, in the GADD45α-GFP Human GreenScreen assay and none induced MN in mouse bone marrow erythrocytes.     

Genotoxicity of gliotoxin,a secondary metabolite of Aspergillus fumigatus,in a battery of short-term test systems

The genotoxic effects of gliotoxin, a known fungal secondary metabolite, were studied. Gliotoxin was purified from cultivation medium of Aspergillus fumigatus isolated from the indoor air of a moisture problem house. The genotoxicity of gliotoxin was assessed both in bacterial test systems including bacterial repair assay, Ames Salmonella assay and SOS-chromotest, and in mammalian cells using single cell gel (SCG) electrophoresis assay and sister-chromatid exchange (SCE) test. Gliotoxin was found to be genotoxic in the bacterial repair assay but, not in the Salmonella test or SOS-chromotest. A dose-related increase in DNA damage was observed in mouse RAW264.7 macrophages exposed to gliotoxin for 2h in plain medium in the SCG assay. In contrast to the positive response in the SCG assay, gliotoxin did not induce any clear, dose-related increase in SCEs in Chinese hamster ovary (CHO) cells.