In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24?h after a single pharyngeal aspiration of MWCNT-S (1–200?μg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2–10.8?mg/m³) for 4 d, 4?h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1–200?μg/mouse) and in MNPCEs after inhalation exposure (17.5?mg/m³). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10?μg/cm²), while MWCNT-T increased strand breakage only at 200?μg/cm². Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.     

The Comet Assay with Multiple Mouse Organs: Comparison of Comet Assay Results and Carcinogenicity with 208 Chemicals Selected from the IARC Monographs and U.S. NTP Carcinogenicity Database

ABSTRACT

The comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity. The purpose of this article is to summarize the in vivo genotoxicity in eight organs of the mouse of 208 chemicals selected from International Agency for Research on Cancer (IARC) Groups 1, 2A, 2B, 3, and 4, and from the U.S. National Toxicology Program (NTP) Carcinogenicity Database, and to discuss the utility of the comet assay in genetic toxicology.

Alkylating agents, amides, aromatic amines, azo compounds, cyclic nitro compounds, hydrazines, halides having reactive halogens, and polycyclic aromatic hydrocarbons were chemicals showing high positive effects in this assay. The responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced by DNA single strand breaks induced chemically and those derived from alkali-labile sites developed from alkylated bases and bulky base adducts. The mouse or rat 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. Therefore, organspecific genotoxicity was necessary but not sufficient for the prediction of organ-specific carcinogenicity. It would be expected that DNA crosslinkers would be difficult to detect by this assay, because of the resulting inhibition of DNA unwinding. The proportion of 10 DNA crosslinkers that was positive, however, was high in the gastrointestinal mucosa, stomach, and colon, but less than 50% in the liver and lung. It was interesting that the genotoxicity of DNA crosslinkers could be detected in the gastrointestinal organs even though the agents were administered intraperitoneally.

Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative nongenotoxic (Ames test-negative) carcinogens. The Ames test is generally used as a first screening method to assess chemical genotoxicity and has provided extensive information on DNA reactivity. Out of 208 chemicals studied, 117 are Ames test-positive rodent carcinogens, 43 are Ames test-negative rodent carcinogens, and 30 are rodent noncarcinogens (which include both Ames test-positive and negative noncarcinogens). High positive response ratio (110/117) for rodent genotoxic carcinogens and a high negative response ratio (6/30) for rodent noncarcinogens were shown in the comet assay. For Ames test-negative rodent carcinogens, less than 50% were positive in the comet assay, suggesting that the assay, which detects DNA lesions, is not suitable for identifying nongenotoxic carcinogens. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. This assay had a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens, suggesting that the comet assay can be used to evaluate the in vivo genotoxicity of in vitro genotoxic chemicals. For chemicals whose in vivo genotoxicity has been tested in multiple organs by the comet assay, published data are summarized with unpublished data and compared with relevant genotoxicity and carcinogenicity data.

Because it is clear that no single test is capable of detecting all relevant genotoxic agents, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. The conventional micronucleus test in the hematopoietic system is a simple method to assess in vivo clastogenicity of chemicals. Its performance is related to whether a chemical reaches the hematopoietic system. Among 208 chemicals studied (including 165 rodent carcinogens), 54 rodents carcinogens do not induce micronuclei in mouse hematopoietic system despite the positive finding with one or two in vitro tests. Forty-nine of 54 rodent carcinogens that do not induce micronuclei were positive in the comet assay, suggesting that the comet assay can be used as a further in vivo test apart from the cytogenetic assays in hematopoietic cells. In this review, we provide one recommendation for the in vivo comet assay protocol based on our own data.     

Oxidative stress and DNA damage in the earthworm <Emphasis Type="Italic">Eisenia fetida</Emphasis> induced by toluene,ethylbenzene and xylene

Superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), and the comet assay (SCGE) were used as biomarkers to evaluate the oxidative stress and genotoxicity of toluene, ethylbenzene and xylene in earthworms (Eisenia fetida). The results indicated that the exposure of the three pollutants caused a stress response of the three enzymes, an approximate bell-shaped change (a tendency of inducement firstly and then inhibition with increasing concentrations of the pollutants) was mostly found. The three enzymes tested differed in their sensitivity to different pollutants. While the activity of POD was not significantly changed within the concentration range, the concentration thresholds for significant (P < 0.05) responses to toluene based on SOD and CAT were 5 mg kg⁻¹, respectively. Similarly, the concentration thresholds for significant (P < 0.05) responses to ethylbenzene based on CAT and POD were 10 and 5 mg kg⁻¹, respectively, while the activity of SOD was not significantly changed within the concentration range. Significant responses to xylene based on CAT and POD were 5 mg kg⁻¹, respectively, while the activity of SOD was significantly (P < 0.05) induced at 10 mg kg⁻¹. The SCGE assay results showed that these three pollutants could significantly (P < 0.01) induce DNA damage in earthworms and the clear dose-dependent relationships were displayed, indicating potential genotoxic effects of toluene, ethylbenzene, and xylene on E. fetida. The inducement of DNA damage may be attributed to the oxidative attack of toluene, ethylbenzene, and xylene. Toluene seemed to be more genotoxic as it could induce the higher extent of DNA damage than ethylbenzene and xylene. The results suggest that the SCGE assay of earthworms is simple and efficient for diagnosing the genotoxicity of pollutants in terrestrial environment.     

Genotoxicity studies on dietary diacylglycerol (DAG) oil.

Dietary diacylglycerol (DAG) oil is an edible oil enriched in DAG (more than 80%). A recent investigation indicated that DAG oil or its components may have beneficial effects on the prevention and management of obesity. We evaluated the genotoxic potential of DAG oil using standard genotoxicity tests. Bacterial reverse mutation assay (Ames test), the chromosomal aberration assay in cultured Chinese hamster lung cells (CHL/IU), and a bone marrow micronucleus assay in ICR CD mice were employed in the present study. In addition we have tested the possibility that genotoxic substances may be formed during cooking, heated DAG oil (HDG) was prepared by batch frying potato slices in the oil at 180 degrees C for 8 h/day for three consecutive days. Therefore, genotoxicity tests were also performed on HDG. Results obtained did not show any genotoxic effect on either unheated DAG oil (UDG) or HDG. We conclude that there are no safety concerns on the genotoxicity of DAG oil under the conditions for normal use.     

The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP Carcinogenicity Database

The comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity. The purpose of this article is to summarize the in vivo genotoxicity in eight organs of the mouse of 208 chemicals selected from International Agency for Research on Cancer (IARC) Groups 1, 2A, 2B, 3, and 4, and from the U.S. National Toxicology Program (NTP) Carcinogenicity Database, and to discuss the utility of the comet assay in genetic toxicology. Alkylating agents, amides, aromatic amines, azo compounds, cyclic nitro compounds, hydrazines, halides having reactive halogens, and polycyclic aromatic hydrocarbons were chemicals showing high positive effects in this assay. The responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced by DNA single strand breaks induced chemically and those derived from alkali-labile sites developed from alkylated bases and bulky base adducts. The mouse or rat 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. Therefore, organ-specific genotoxicity was necessary but not sufficient for the prediction of organ-specific carcinogenicity. It would be expected that DNA crosslinkers would be difficult to detect by this assay, because of the resulting inhibition of DNA unwinding. The proportion of 10 DNA crosslinkers that was positive, however, was high in the gastrointestinal mucosa, stomach, and colon, but less than 50% in the liver and lung. It was interesting that the genotoxicity of DNA crosslinkers could be detected in the gastrointestinal organs even though the agents were administered intraperitoneally. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative nongenotoxic (Ames test-negative) carcinogens. The Ames test is generally used as a first screening method to assess chemical genotoxicity and has provided extensive information on DNA reactivity. Out of 208 chemicals studied, 117 are Ames test-positive rodent carcinogens, 43 are Ames test-negative rodent carcinogens, and 30 are rodent noncarcinogens (which include both Ames test-positive and negative noncarcinogens). High positive response ratio (110/117) for rodent genotoxic carcinogens and a high negative response ratio (6/30) for rodent noncarcinogens were shown in the comet assay. For Ames test-negative rodent carcinogens, less than 50% were positive in the comet assay, suggesting that the assay, which detects DNA lesions, is not suitable for identifying nongenotoxic carcinogens. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. This assay had a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens, suggesting that the comet assay can be used to evaluate the in vivo genotoxicity of in vitro genotoxic chemicals. For chemicals whose in vivo genotoxicity has been tested in multiple organs by the comet assay, published data are summarized with unpublished data and compared with relevant genotoxicity and carcinogenicity data. Because it is clear that no single test is capable of detecting all relevant genotoxic agents, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. The conventional micronucleus test in the hematopoietic system is a simple method to assess in vivo clastogenicity of chemicals. Its performance is related to whether a chemical reaches the hematopoietic system. Among 208 chemicals studied (including 165 rodent carcinogens), 54 rodents carcinogens do not induce micronuclei in mouse hematopoietic system despite the positive finding with one or two in vitro tests. Forty-nine of 54 rodent carcinogens that do not induce micronuclei were positive in the comet assay, suggesting that the comet assay can be used as a further in vivo test apart from the cytogenetic assays in hematopoietic cells. In this review, we provide one recommendation for the in vivo comet assay protocol based on our own data.     

Study of the cytotoxic and genotoxic potential of the carbonyl ruthenium(II) compound,ct‐[RuCl(CO)(dppb)(bipy)]PF6 [dppb = 1,4‐bis(diphenylphosphino)butane and bipy = 2,2′‐bipyridine], by in vitro and in vivo assays

Considering the promising previous results of ct‐[RuCl(CO)(dppb)(bipy)]PF₆ (where dppb = 1,4‐bis(diphenylphosphino)butane and bipy = 2,2′‐bipyridine) as an antitumor agent, novel biological assays evaluating its toxicogenic potential were performed. The genotoxicity of the compound was evaluated by the in vitro micronucleus test (V79, Chinese hamster lung fibroblasts; HepG2, hepatocellular carcinoma cells), in vivo bone marrow micronucleus test and comet assay in hepatocytes (Swiss mice). The animals were treated with 0.63, 1.25, 2.5 and 5.0 mg/kg body weight (bw) of the compound. Negative (water) and positive (cisplatin, 1.5 mg/kg bw; methyl methanesulfonate, 40 mg/kg bw) controls were included. The parameters considered in the comet assay were the percentage of tail DNA, tail moment and tail length. The results of the in vitro micronucleus tests showed the absence of genotoxicity in V79 cells, while the compound was genotoxic in HepG2 cells at a concentration of 1.25 μm . In the in vivo micronucleus test, the compound was not genotoxic at the different doses evaluated. In the comet assay, only the dose of 5.0 mg/kg bw resulted in a significant increase in the frequency of DNA damage in hepatocytes when compared to the negative control. The genotoxic effect observed in HepG2 cells and in the liver comet assay indicates that the compound was metabolized by hepatic cells.     

Evaluation of the genotoxic potential of single-wall carbon nanotubes by using a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of a high purity sample of single-wall carbon nanotubes (SWCNTs) was evaluated using a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse mutation test (Ames test), an in vitro chromosomal aberration test, and an in vivo mouse bone marrow micronucleus test. The SWCNTs exerted no genotoxicity in Salmonella typhimurium TA97, TA98, TA100, and TA1535, or in Escherichia coli WP2 uvrA/pKM101, whether in the absence or presence of metabolic activation and at concentrations of 12.5–500 μg/plate. In the chromosomal aberration test, at 300–1000 μg/mL, the SWCNTs did not increase the number of structural or numerical chromosomal aberrations, whether the test was conducted with or without metabolic activation. In the in vivo bone marrow micronucleus test, doses of 60 mg/kg and 200 mg/kg SWCNTs did not affect the proportions of immature and total erythrocytes, nor did it increase the number of micronuclei in the immature erythrocytes of mice. The results of these studies show that the high purity and well-dispersed sample of SWCNTs are not genotoxic under the conditions of the in vitro bacterial reverse mutation assay, chromosomal aberration assay, or in vivo bone marrow micronucleus test, and thus appear not to pose a genotoxic risk to human health in vivo.     

Genotoxicity studies on green tea catechin

The beneficial effects of tea catechins are well documented. We evaluated the genotoxic potential of a green tea catechin preparation using established genotoxicity assays, including a bacterial reverse mutation assay (Ames test), a chromosomal aberration assay in cultured Chinese hamster lung cells (CHL/IU), a mouse lymphoma L5178Y/tk assay, and a bone marrow micronucleus (MN) assay in ICR CD mice and SD rats. No significant increases in the number of revertant colonies were observed in the Ames test, but positive responses were observed in two in vitro assays: the chromosomal aberration assay and mouse lymphoma L5178/tk assay. However, the in vivo study demonstrated no significant increase in micronucleated polychromatic erythrocytes (MNPCE) in the bone marrow of both ICR CD mice and SD rats administered a high dose of the green tea catechin preparation up to 2000 mg/kg. Combined with favorable epidemiological information suggesting a chemopreventive effect of tea catechins on carcinogenesis, we conclude that green tea catechin presents no significant genotoxic concern under the anticipated conditions of use. These results are consistent with other genotoxicity studies of tea catechins, which show minimal, if any, genotoxic potential.     

Genotoxicity evaluation of dimethoate to experimental mice by micronucleus, chromosome aberration tests, and comet assay

Dimethoate (DM) is an organophosphate insecticide with numerous uses on field and agricultural crops and ornamentals. Data concerning DM-acute genotoxicity are controversial and knowledge on its delayed effect is limited. For this reason, we aimed to further explore DM genotoxicity resulting from subchronic intoxication of experimental mice. Thus, DM was administered to mice at doses ranging from 1 to 30 mg/kg body weight for a period of 30 consecutive days. There was a significant increase (P < .05) in the frequency of micronucleated bone marrow cells following DM administration. Furthermore, the chromosome aberration assay revealed a significant increase in the percentage of chromosome abnormalities in a dose-dependent manner. Dimethoate was also found to induce significant DNA damage in mouse bone marrow cells as assessed by the comet assay. Altogether, our results showed that, after a subchronic exposure, DM was a genotoxic compound in experimental mice.     

Genetic toxicity of naphthalene: a review

Results of five previously unpublished studies of the genotoxicity of naphthalene are presented and extensively discussed in relation to the large database that exists in the published literature. According to the published literature, naphthalene has not induced gene mutations in bacterial assays or in a metabolically competent human cell line. However, naphthalene has caused cytotoxicity in some cell lines, and induced clastogenicity in Chinese hamster ovary (CHO) cells, in a human lymphoblastoid cell line, and in preimplantation mouse embryos. Some naphthalene metabolites were cytotoxic, but only naphthoquinones produced chromosomal damage in vitro. No chromosomal damage was observed in vivo in bone marrow erythrocytes from treated mice; however, a positive response was reported in a Drosophila assay for wing somatic mutation and recombination. The five unpublished studies of naphthalene genotoxicity include three studies in vitro (two Ames bacterial assays and an in vitro unscheduled DNA synthesis assay) and two in vivo (mouse micronucleus and in vivo unscheduled DNA synthesis). Naphthalene was inactive in all five studies, in agreement with reports in the published literature. Chronic inhalation of naphthalene over 2 yr induced an increased incidence of benign alveolar/bronchial adenomas in female mice, and nasal epithelial tumors in both sexes of rats. Inflammation, tissue damage, and subsequent regenerative hyperplasia at target organ sites occurred in both species. Results of standard genetic toxicity assays suggest that naphthalene is not likely to be genotoxic in vivo. Since the in vitro results come primarily from assays utilizing liver-mediated activation systems, and the in vivo results come from rodent organs that are not targets for tumors, tests using naphthalene-sensitive rodent tissues would determine the applicability of current data in addressing the mechanisms of these species and site-specific cancers. The standard assays reported here may be useful in predicting potential health hazard in other species, or in humans, in whom there are few reported instances of naphthalene-induced cancer, especially as more data on species-specific differences in naphthalene metabolism become available. Despite present data limitations, a threshold mechanism for tumorigenesis can be proposed. The absence of naphthalene-induced gene mutation and the presence of cytotoxicity and some chromosomal events in vitro are consistent with a threshold-related mechanism of tumor induction, driven by cytotoxicity and cell regeneration, followed by genetic events, or by accumulation of naphthalene at specific target sites to allow in situ formation of a genotoxic metabolite to trigger or enhance spontaneous tumor development.     

Inhibition of cytochrome P450 2E1 decreases, but does not eliminate, genotoxicity mediated by 1,3-butadiene.

1,3-Butadiene (BD), a rodent carcinogen, is metabolized to mutagenic and DNA-reactive epoxides. In vitro data suggest that this oxidation is mediated by cytochrome P450 2E1 (CYP2E1). In this study, we tested the hypothesis that oxidation of BD by CYP2E1 is required for genotoxicity to occur. Inhalation exposures were conducted with B6C3F1 mice using a closed-chamber technique, and the maximum rate of butadiene oxidation was estimated. The total amount of butadiene metabolized was then correlated with the frequency of micronuclei (MN). Three treatment groups were used: (1) mice with no pretreatment; (2) mice pretreated with 1,2-trans-dichloroethylene (DCE), a specific CYP2E1 inhibitor; and (3) mice pretreated with 1-aminobenzotriazole (ABT), an irreversible inhibitor of cytochromes P450. Mice in all 3 groups were exposed to an initial BD concentration of 1100 ppm, and the decline in concentration of BD in the inhalation chamber with time, due to uptake and metabolism of BD, was monitored using gas chromatography. A physiologically based pharmacokinetic model was used to analyze the gas uptake data, estimate V(max) for BD oxidation, and compute the total amount of BD metabolized. Model simulations of the gas uptake data predicted the maximum rate of BD oxidation would be reduced by 60% and 100% for the DCE- and ABT-pretreated groups, respectively. Bone marrow was harvested 24 h after the onset of the inhalation exposure and analyzed for frequency of micronuclei in polychromatic erythrocytes (MN-PCE). The frequency of MN-PCE per 1000 PCE in mice exposed to BD was 28.2 +/- 3.1, 19.8 +/- 2.5, and 12.3 +/- 1.9, for the mice with no pretreatment, DCE-pretreated mice and ABT-pretreated mice, respectively. Although inhibition of CYP2E1 decreased BD-mediated genotoxicity, it did not completely eliminate genotoxic effects. These data suggest that other P450 isoforms may contribute significantly to the metabolic activation of BD and resultant genotoxicity.     

Genotoxicity hazard assessment of Caramel Colours III and IV.

Results from a battery of short-term tests in vitro and in vivo used to assess the genotoxicity of caramel colours are presented and discussed in relation to reports from the literature. No evidence of genotoxicity was found in the Salmonella plate incorporation test using five standard strains or in the Saccharomyces cerevisiae gene conversion assay using strain D4, either with or without S-9 for activation. A weak clastogenic effect for a sample of Caramel Colour III in CHO cells was abolished in the presence of S-9. Two samples of Caramel Colour IV were not clastogenic in CHO cells. Salmonella pre-incubation tests without S-9 also failed to reveal any mutagenic activity for any of the caramel colours tested. The Caramel Colour III sample that showed clastogenic activity in CHO cells in vitro did not induce micronuclei when evaluated in a mouse bone marrow assay. These results are in general agreement with reports in the literature regarding the genotoxicity of caramel colours, and support the conclusion that caramel colours do not pose a genotoxic hazard to humans.     

Genotoxicity of pyrroloquinoline quinone (PQQ) disodium salt (BioPQQ™)

The genotoxic potential of pyrroloquinoline quinone (PQQ) disodium salt (BioPQQ™) was evaluated in a battery of genotoxicity tests. The results of the bacterial mutation assay (Ames test) were negative. Weak positive results were obtained in 2 separate in vitro chromosomal aberration test in Chinese hamster lung (CHL) fibroblasts. Upon testing in an in vitro chromosomal aberration test in human peripheral blood lymphocytes, no genotoxic activity of PQQ was noted. In the in vivo micronucleus assay in mice, PQQ at doses up to 2000 mg/kg body weight demonstrated that no genotoxic effects are expressed in vivo in bone marrow erythrocytes. The weak responses in the in vitro test CHL cells were considered of little relevance under conditions of likely human exposure. PQQ disodium was concluded to have no genotoxic activity in vivo.     

Oxidative stress and DNA damage responses in rat and mouse lung to inhaled carbon nanoparticles

Abstract

We have investigated whether short-term nose-only inhalation exposure to electric spark discharge-generated carbon nanoparticles (～60 nm) causes oxidative stress and DNA damage responses in the lungs of rats (152 μg/m³; 4 h) and mice (142 μg/m³; 4 h, or three times 4 h). In both species, no pulmonary inflammation and toxicity were detected by bronchoalveolar lavage or mRNA expression analyses. Oxidative DNA damage (measured by fpg-comet assay), was also not increased in mouse whole lung tissue or isolated lung epithelial cells from rat. In addition, the mRNA expressions of the DNA base excision repair genes OGG1, DNA Polβ and XRCC1 were not altered. However, in the lung epithelial cells isolated from the nanoparticle-exposed rats a small but significant increase in APE-1 mRNA expression was measured. Thus, short-term inhalation of carbon nanoparticles under the applied exposure regimen, does not cause oxidative stress and DNA damage in the lungs of healthy mice and rats.     

In vivo protective effect of Uridine,a pyrimidine nucleoside,on genotoxicity induced by Levodopa/Carbidopa in mice

Parkinson's disease (PD) is a common neurodegenerative disorder that affects millions of people all over the world. Motor symptoms of PD are most commonly controlled by L-3,4-dihydroxyphenylalanine (Levodopa, L-DOPA), a precursor of dopamine, plus a peripherally-acting aromatic-L-amino-acid decarboxylase (dopa decarboxylase) inhibitor, such as carbidopa. However, chronic treatment with a combination of Levodopa plus carbidopa has been demonstrated to cause a major complication, namely abnormal involuntary movements. On the other hand, the effect of this treatment on bone marrow cells is unknown. Therefore, in this study, we aimed to investigate possible genotoxic effects of Levodopa and Carbidopa using male Balb/C mice. Our results showed that Levodopa alone or in combination with carbidopa caused genotoxicity in in vivo micronucleus test (mouse bone marrow) and Comet assay (blood cells). Furthermore, we showed that simultaneous administration of uridine, a pyrimidine nucleoside, reversed the genotoxic effect of Levodopa and Carbidopa in both assays. Our data show for the first time that Levodopa plus carbidopa combination causes genotoxicity which is reversed by uridine treatment. These findings might enhance our understanding for the complications of a common Parkinson's treatment and confer benefit in terms of reducing a possible genotoxic effect of this treatment.     

Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles

Engineered nanoparticles (NPs) are widely used in different technologies but their unique properties might also cause adverse health effects. In reviewing recent in vitro and in vivo genotoxicity studies we discuss potential mechanisms of genotoxicity induced by NPs. Various factors that may influence genotoxic response, including physico-chemical properties and experimental conditions, are highlighted. From 4346 articles on NP toxicity, 112 describe genotoxicity studies (94 in vitro, 22 in vivo). The most used assays are the comet assay (58 in vitro, 9 in vivo), the micronucleus assay (31 in vitro, 14 in vivo), the chromosome aberrations test (10 in vitro, 1 in vivo) and the bacterial reverse mutation assay (13 studies). We describe advantages and potential problems with different methods and suggest the need for appropriate methodologies to be used for investigation of genotoxic effects of NPs, in vitro and in vivo.     

聚乙二醇修饰降纤酶的遗传毒性评价

目的检测聚乙二醇修饰降纤酶的遗传毒性。方法应用鼠伤寒沙门菌回复突变试验(Ames试验)、体外培养CHO细胞染色体畸变试验和小鼠骨髓微核试验检测聚乙二醇修饰降纤酶的遗传毒性。结果 Ames试验结果显示每平皿100、20、4、0.8、0.16 U各个剂量组,在加或不加S9代谢活化系统时对组氨酸缺陷型鼠伤寒沙门菌TA97、TA98、TA100、TA102及TA1535所诱发的回复突变菌落数均与溶剂对照的突变菌落数相近。体外培养CHO细胞染色体畸变试验结果显示2.5、5.0和10.0 U.mL-1各个剂量组在加S9代谢活化系统于24 h和不加S9代谢活化系统于24 h、48 h培养的CHO细胞染色体畸变率与溶剂对照组比较均无显著差异(P>0.05)。小鼠骨髓微核试验显示425、850、1700 U.kg-1各个剂量组对ICR小鼠的微核诱发率与溶剂对照组比较均无显著差异(P>0.05)。结论聚乙二醇修饰降纤酶对鼠伤寒沙门菌无致突变性,对哺乳动物培养细胞的染色体无致畸变作用,对ICR小鼠无诱发骨髓嗜多染红细胞微核的效应。表明聚乙二醇修饰降纤酶在本实验条件下无遗传毒性。     

Evaluation of genotoxicity of oral exposure to tetravalent vanadium in vivo

The trace element vanadium interacts with living cells, in which it exerts a variety of biological effects depending on its chemical form and oxidation state. Tetravalent vanadium was shown to affect several genotoxicity end-points in vitro, but its genotoxic potential in vivo is not elucidated. In this study, the genotoxic effects induced in vivo by subacute oral exposure to vanadyl sulphate (VOSO4), a tetravalent vanadium salt, were investigated. To this aim male CD1 mice were administered with VOSO4 in drinking water over the dose range 2-1000 mg/l for 5 weeks. The incidence of micronucleated blood reticulocytes was measured along treatment period. At the end of treatment, micronuclei in both blood reticulocytes and bone marrow polychromatic erythrocytes were determined; in addition, DNA lesions detectable by comet assay were assessed in marrow and testicular cells. Tissue distribution of vanadium at sacrifice was determined by atomic absorption spectrometry. Comet assays and the analysis of micronuclei in polychromatic erythrocytes did not reveal treatment related effects. A slight increase in micronucleated reticulocytes, with no relationship with the administered dose, was observed in some treated groups. The determination of vanadium content in kidney, liver, spleen, bone, stomach, small intestine and testis highlighted low internal exposure, especially in soft tissues. Overall, data indicate scarce bioavailability for orally administered tetravalent vanadium, and lack of significant genotoxic potential in vivo.     

Hemangiosarcoma in mice administered pregabalin: analysis of genotoxicity, tumor incidence, and tumor genetics

Pregabalin, (S)-3-(aminomethyl)-5-methylhexanoic acid, binds with high affinity to the α(2)δ subunit of voltage-gated calcium channels and exerts analgesic, anxiolytic, and antiseizure activities. Two-year carcinogenicity studies were completed in B6C3F1 and CD-1 mice and two separate studies in Wistar rats. Doses in mice were 200, 1000, and 5000 mg/kg/day, with systemic exposures (AUC(0-24 h)) up to 31 times the mean exposure in humans, given the maximum recommended clinical dose. In rats, doses were 50, 150, and 450 mg/kg/day in males and 100, 300, and 900 mg/kg/day in females; systemic exposures up to 24 times were achieved in clinical trials. In both strains of mice, pregabalin treatment was associated with an increased incidence of hemangiosarcoma primarily in liver, spleen, and bone marrow. The incidence of hemangiosarcoma was higher in B6C3F1 mice than in CD-1 mice, consistent with its spontaneous incidence. Pregabalin did not increase the incidence of any other tumor type in rats and was not genotoxic, based on an extensive battery of in vivo and in vitro tests in bacterial and mammalian systems. Thus, pregabalin is a single-species, single tumor-type, nongenotoxic mouse carcinogen. Hemangiosarcomas occurring in mice treated with pregabalin were genotypically distinct from hemangiosarcomas induced by genotoxic carcinogens in humans with respect to ras and p53 mutation patterns and were similar to spontaneous tumors. Furthermore, there was a strong association between pregabalin treatment and bone marrow changes in these studies in mice, suggesting a possible link between the effects observed in bone marrow and the increase in tumor incidence in pregabalin-treated mice.     

Relative sensitivity of fish and mammalian cells to the antibiotic, trimethoprim: cytotoxic and genotoxic responses as determined by neutral red retention, Comet and micronucleus assays

Relative cytotoxicity and genotoxicity of a widely used antibiotic, trimethoprim (TRIMP) was evaluated under in vitro conditions using rainbow trout gonad-2 (RTG-2) and Chinese hamster ovary-K1 (CHO-K1) cells. Whilst cytotoxicity was determined using neutral red retention (NRR) assay, the genotoxicity was determined using single cell gel electrophoresis or the Comet assay and cytokinesis-block micronucleus (CBMN) assay. For NRR assay, concentration-dependent cytotoxic effect was observed for both the cell lines (estimated EC₅₀ values: 671.82 ± 21.78 and 611.6 ± 20.4 μg ml⁻¹ for RTG-2 and CHO-K1 cells, respectively). There was no statistically significant difference between the two cell lines for this assay. For the Comet assay, standard 6 h exposure to TRIMP did not show any positive response for any of the cell types used. However, 48 h exposure to RTG-2 cells showed a concentration-dependent induction of DNA damage (r = 0.86). The highest concentration of TRIMP used (i.e. 100 μg ml⁻¹) showed relatively higher DNA damage, compared to ethyl methane sulfonate (EMS; 1 μg ml⁻¹ or 8 mM), a reference genotoxic agent, used concurrently. In contrast, 24 h exposure time for CHO-K1 cells did not show any concentration-dependent increase for this assay. For MN assay, a significant correlation was found between the MN induction and TRIMP concentration for both the cell lines (RTG-2: r = 0.68; CHO-K1: r = 0.79), although only the highest concentration used showed a significant increase for binucleated (BN) cell with micronuclei (BNMN). The study suggests that whilst the cells of different origin could exhibit similar cytotoxicity, they could display differential genotoxic effects. Furthermore, genotoxic effects of TRIMP are primarily exposure period dependent phenomena and, in addition to inhibiting the action of dihydrofolate reductase, oxidative stress could also contribute for the observed toxic effects, fish cells in general being more sensitive for genotoxic effects.