Primary DNA Damage Research Articles

Terrestrial gastropods (Helix spp) as sentinels of primary DNA damage for biomonitoring purposes: A validation study

We validated the alkaline comet assay in two species of land snail (Helix aspersa and Helix vermiculata) to test their suitability as sentinels for primary DNA damage in polluted environments. The study was conducted under the framework of a biomonitoring program for a power station in Central Italy that had recently been converted from oil to coal-fired plant. After optimizing test conditions, the comet assay was used to measure the % Tail DNA induced by in vitro exposure of hemocytes to different concentrations of a reactive oxygen species (H2 O2 ). The treatment induced significant increases in this parameter with a concentration effect, indicating the effectiveness of the assay in snail hemocytes. After evaluating possible differences between the two species, we sampled them in three field sites at different distances from the power station, and in two reference sites assumed to have low or no levels of pollution. No species differences emerged. Percent Tail DNA values in snails from the sites near the power station were higher than those from control sites. An inverse correlation emerged between % Tail DNA and distance from the power station, suggesting that the primary DNA damage decreased as distance increased away from the pollution source. Detection of a gradient of heavy metal concentration in snail tissues suggests that these pollutants are a potential cause of the observed pattern. The comet assay appears to be a suitable assay and Helix spp. populations suitable sentinels to detect the genotoxic impact of pollutants.

Ex vivo evaluation of genotoxic effects of four dental adhesives on human leukocytes

Background/purposeThe use of dental adhesives in everyday dental practice has raised questions about their biologic safety. Their biocompatibility is a relevant aspect of the clinical success of these materials. The objective of this study was to evaluate the genotoxicity of dental adhesives ex vivo using a cytogenetic assay. Materials and methodsFour materials (AdheSE, G-Bond, Excite, and Adper Single Bond 2) were tested on human peripheral blood leukocytes using a comet assay. Prepared materials were eluted in a saline solution for 1 hour, 1 day, and 5 days. The comet assay was used to evaluate primary DNA damage by measuring the tail length and tail intensity. A Kruskall-Wallis nonparametric test was used for the statistical analysis, with the significance level set to P < 0.05. ResultsNone of the tested dental adhesives revealed a statistically significant increase in the tail length or tail intensity in treated leukocytes, independent of the applied dilution, elution duration, and polymerization form. A slight increase in the tail length and intensity of DNA molecules was observed after 1 and 5 days of the elution period at the lowest dilution (1:102) for all tested adhesives, only in their nonpolymerized form; however, these results were not statistically significant. ConclusionUnder the conditions used in this study, all adhesives had acceptable biocompatibility in terms of genotoxicity.

What is Comet assay not telling us: AFLP reveals wider aspects of genotoxicity

DNA damage detected by genotoxicity biomarkers such as the Comet assay is not always a reliable indicator of the consequences that genotoxic agents can have on the genome integrity of the exposed organisms. Therefore, to reveal the existence of more permanent alterations of DNA structure after genotoxic stress, the RTG-2 rainbow trout cell line was exposed for 3days to benzo[a]pyrene (B[a]P, 0.1–10μM) and ethyl methanesulfonate (EMS, 0.1–1mM) followed by 3days of recovery period. Primary DNA damage was evaluated by the Comet assay and DNA alterations were assessed using AFLP (amplified fragment length polymorphism). Qualitative and quantitative modifications in AFLP profiles were analyzed in order to detect genetic alterations arising from mutation events and/or DNA damage. Significant induction in DNA damage measured by the Comet assay was noticed after B[a]P treatment at all concentrations but values returned to the control level after recovery. Exposure to EMS induced significant DNA damage only at the highest concentration and damage persisted after the recovery period. AFLP profiles detected DNA alterations even when Comet assay indicated complete DNA repair, revealing more persistent damage. Since such DNA damage can impair its structure and function, Comet assay results should preferably be supplemented with other methods in order to predict the consequences of genotoxic insult more accurately.

Differential responses of sexual and asexual Artemia to genotoxicity by a reference mutagen: Is the comet assay a reliable predictor of population level responses?

The impact of chronic genotoxicity to natural populations is always questioned due to their reproductive surplus. We used a comet assay to quantify primary DNA damage after exposure to a reference mutagen ethyl methane sulfonate in two species of crustacean with different reproductive strategies (sexual Artemia franciscana and asexual Artemia parthenogenetica). We then assessed whether this predicted individual performance and population growth rate over three generations. Artemia were exposed to different chronic concentrations (0.78mM, 1.01mM, 1.24mM and 1.48mM) of ethyl methane sulfonate from instar 1 onwards for 3 h, 24 h, 7 days, 14 days and 21 days and percentage tail DNA values were used for comparisons between species. The percentage tail DNA values showed consistently elevated values up to 7 days and showed a reduction from 14 days onwards in A. franciscana. Whilst in A. parthenogenetica such a reduction was evident on 21 days assessment. The values of percentage tail DNA after 21 days were compared with population level fitness parameters, growth, survival, fecundity and population growth rate to know whether primary DNA damage as measured by comet assay is a reliable biomarker. Substantial increase in tail DNA values was associated with substantial reductions in all the fitness parameters in the parental generation of A. franciscana and parental, F1 and F2 generations of A. parthenogenetica. So comet results were more predictive in asexual species over generations. These results pointed to the importance of predicting biomarker responses from multigenerational consequences considering life history traits and reproductive strategies in ecological risk assessments.

Genotoxicity in gingival cells of patients undergoing tooth restoration with two different dental composite materials

Dental composite materials come into direct contact with oral tissue, especially gingival cells. This study was performed to evaluate possible DNA damage to gingival cells exposed to resin composite dental materials. Class V restorations were placed in 30 adult patients using two different composite resins. The epithelial cells of the gingival area along the composite restoration were sampled prior to and after 7, 30, and 180days following the restoration of the tooth. DNA damage was analysed by comet and micronucleus assays in gingival exfoliated epithelial cells. The results showed significantly higher comet assay parameters (tail length and % DNA in the tail) within periods of 30 and 180days. The micronucleus test for the same exposure time demonstrated a higher number of cells with micronuclei, karyolysis, and nuclear buds. Results did not reveal any difference between the two composite materials for the same duration of exposure. Based on the results, we can conclude that the use of composite resins causes cellular damage. As dental composite resins remain in intimate contact with oral tissue over a long period of time, further research on their possible genotoxicity is advisable. Long-term exposure of gingival cells to two different composite materials demonstrated certain DNA damage. However, considering the significant decline in micronuclei frequency after 180days and efficiency in the repair of primary DNA damage, the observed effects could not be indicated as biologically relevant.

UPLC-TOF-MS Characterization and Identification of Bioactive Iridoids inCornus masFruit

Cornus mas L. is indigenous to Europe and parts of Asia. Although Cornus is widely considered to be an iridoid rich genera, only two iridoids have been previously found in this plant. The lack of information on taxonomically and biologically active iridoids prompted us to develop and optimize an analytical method for characterization of additional phytochemicals in C. mas fruit. An ultra performance liquid chromatography (UPLC) coupled with photodiode array spectrophotometry (PDA) and electrospray time-of-flight mass spectrometry (ESI-TOF-MS) was employed and mass parameters were optimized. Identification was made by elucidating the mass spectral data and further confirmed by comparing retention times and UV spectra of target peaks with those of reference compounds. Primary DNA damage and antigenotoxicity tests in E. coli PQ37 were used to screen the iridoids for biological activity. As a result, ten phytochemicals were identified, including iridoids loganic acid, loganin, sweroside, and cornuside. Nine of these were reported for the first time from C. mas fruit. The iridoids did not induce SOS repair of DNA, indicating a lack of genotoxic activity in E. coli PQ37. However, loganin, sweroside, and cornuside did reduce the amount of DNA damage caused by 4-nitroquinoline 1-oxide, suggesting potential antigenotoxic activity.

Electrochemical genotoxicity assay based on a SOS/umu test using hydrodynamic voltammetry in a droplet.

The SOS/umu genotoxicity assay evaluates the primary DNA damage caused by chemicals from the β-galactosidase activity of S. typhimurium. One of the weaknesses of the common umu test system based on spectrophotometric detection is that it is unable to measure samples containing a high concentration of colored dissolved organic matters, sediment, and suspended solids. However, umu tests with electrochemical detection techniques prove to be a better strategy because it causes less interference, enables the analysis of turbid samples and allows detection even in small volumes without loss of sensitivity. Based on this understanding, we aim to develop a new umu test system with hydrodynamic chronoamperometry using a rotating disk electrode (RDE) in a microliter droplet. PAPG when used as a substrate is not electroactive at the potential at which PAP is oxidized to p-quinone imine (PQI), so the current response of chronoamperometry resulting from the oxidation of PAP to PQI is directly proportional to the enzymatic activity of S. typhimurium. This was achieved by performing genotoxicity tests for 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2) and 2-aminoanthracene (2-AA) as model genotoxic compounds. The results obtained in this study indicated that the signal detection in the genotoxicity assay based on hydrodynamic voltammetry was less influenced by the presence of colored components and sediment particles in the samples when compared to the usual colorimetric signal detection. The influence caused by the presence of humic acids (HAs) and artificial sediment on the genotoxic property of selected model compounds such as 4-nitroquinoline-N-oxide (4-NQO), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), 1,8-dinitropyrene (1,8-DNP) and 1-nitropyrene (1-NP) were also investigated. The results showed that the genotoxicity of 1-NP and MX changed in the presence of 10 mg·L−1 HAs. The genotoxicity of tested chemicals with a high hydrophobicity such as 1,8-DNP and 1-NP were decreased substantially with the presence of 1 g·L−1 sediment. This was not observed in the case of genotoxins with a low log Kow value.

Assessment of the adverse effects of the acaricide amitraz: in vitro evaluation of genotoxicity

Amitraz is a formamidine widely used in Veterinary Medicine for the treatment of ectoparasites. It is a highly liposoluble compound that is quickly absorbed through the skin and mucous membranes, thus making exposure potentially dangerous for humans and animals. The aim of this study was to compare the genotoxic potential of the active constituent of the insecticide amitraz and a commercial product containing amitraz in vitro in hamster cells. The induction of primary DNA damage was evaluated by alkaline single-cell gel electrophoresis (comet assay) and the apoptotic ability was examined by the Annexin V/propidium iodide staining assay. The commercial formulation significantly increased the index of DNA damage at concentrations of 2.50–3.75 µg/mL compared to the control. The active constituent only induced significant DNA damage with the highest concentration (3.75 µg/mL). Although both tested products increased the frequency of cell death, neither of them induced significant differences. Genotoxic potential is a primary risk factor for long-term effects such as carcinogenic and reproductive toxicology. Results presented here highlight the need for further investigation of the potential health risk of this veterinary medicine.

Cytogenetic analyses of Azadirachtin reveal absence of genotoxicity but marked antiproliferative effects in human lymphocytes and CHO cells in vitro

In this work we have examined the genotoxic potential of the bioinsecticide Azadirachtin A (AZA) and its influence on cell proliferation on human lymphocytes and Chinese Hamster ovary (CHO) cells. AZA genotoxicity was assessed by the analysis of chromosomal aberrations and sister chromatid exchanges (SCEs) in the absence and presence of rat liver S9 metabolism. Primary DNA damage was also investigated by means of the comet assay. The results obtained clearly indicate that AZA is not genotoxic in mammalian cells. On the other hand, AZA proved to interfere with cell cycle progression as shown by modulation of frequencies of first (M1) and second division (M2) metaphases detected by 5-Bromo-2′-deoxyuridine labeling. Accumulation of M1 metaphases were more pronounced in human lymphocytes. In the transformed CHO cell line, however, significant increases of multinucleated interphases and polyploid cells were observed at long treatment time. At higher dose-levels, the incidence of polyploidy was close to 100%. Identification of spindle structure and number of centrosomes by fluorescent immunostaining with α- and γ-tubulin antibodies revealed aberrant mitoses exhibiting multipolar spindles with several centrosomal signals.These findings suggest that AZA can act either through a stabilizing activity of microtubules or by inhibition of Aurora A, since both mechanisms are able to generate genetically unstable polyploid cells with multipolar spindles and multinucleated interphases.

Hepatoprotective potential of astaxanthin against 2,3,7,8-tetrachlorodibenzo-p-dioxin in cultured rat hepatocytes

The purpose of this study was to evaluate the effect of carotenoid astaxanthin (ASTA) on cultured primary rat hepatocytes treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the cell viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT), lactate dehydrogenase (LDH) activity, 8-oxo-2-deoxyguanosine (8-OH-dG), total antioxidant capacity (TAC), and total oxidative stress (TOS) levels, and liver micronucleus rates. ASTA (2.5, 5, and 10 µM) was added to cultures alone or simultaneously with TCDD (5 and 10 µM) for 48 h. The results of MTT and LDH assays showed that both doses of TCDD caused significant decrease in cell viability. Also, TCDD significantly increased TOS and decreased TAC level in rat hepatocytes. On the basis of increasing doses, the dioxin caused significant increase in micronucleated hepatocytes) and 8-OH-dG level as compared to control culture. The presence of ASTA with TCDD minimized its effects on primary hepatocytes cultures and DNA damages.

Evaluation of genotoxicity and DNA protective effects of mangiferin, a glucosylxanthone isolated from Mangifera indica L. stem bark extract

Mangiferin is a glucosylxantone isolated from Mangifera indica L. stem bark. Several studies have shown its pharmacological properties which make it a promising candidate for putative therapeutic use. This study was focused to investigate the in vitro genotoxic effects of mangiferin in the Ames test, SOS Chromotest and Comet assay. The genotoxic effects in bone marrow erythrocytes from NMRI mice orally treated with mangiferin (2000mg/kg) were also evaluated. Additionally, its potential antimutagenic activity against several mutagens in the Ames test and its effects on CYP1A1 activity were assessed. Mangiferin (50–5000μg/plate) did not increased the frequency of reverse mutations in the Ames test, nor induced primary DNA damage (5–1000μg/mL) to Escherichia coli PQ37 cells under the SOS Chromotest. It was observed neither single strand breaks nor alkali-labile sites in blood peripheral lymphocytes or hepatocytes after 1h exposition to 10–500μg/mL of mangiferin under the Comet assay. Furthermore, micronucleus studies showed mangiferin neither induced cytotoxic activity nor increased the frequency of micronucleated/binucleated cells in mice bone marrow. In short, mangiferin did not induce cytotoxic or genotoxic effects but it protect against DNA damage which would be associated with its antioxidant properties and its capacity to inhibit CYP enzymes.

Biomarkers of mild hyperthermia related to flashover training in firefighters

Flashover phenomenon occurs when surfaces exposed to thermal radiation reach the ignition temperature, and the fire rapidly spreads in enclosed area. Flashover training (FOT) performed by firefighters is a simulation of flashover phenomenon under controlled conditions. The study aimed to test thermal and physical strain in male firefighters and instructors attending FOT and its influence on DNA damage, exhaled breath condensate (EBC) pH, and fraction of exhaled nitric oxide (FeNO). DNA damage markers were analyzed in 51 attendees and 7 instructors, and EBC pH and FeNO in 40 respiratory healthy non-smoking subjects (34 attendees and 6 instructors).The average body temperature and pulse increase was 1.1°C and 30beats per minute, respectively. A prominent increase in the alkali-labile sites' level has been observed in instructors' peripheral leukocytes compared to first-time attendees (tail length p=0.050, % of DNA in tail p=0.005). FOT was related only to physiological FVC and FEV1 increase (by 4% and 2.7% on average), and FeNO dropped after the exercise by 2ppb in comparison with basal values (P=0.034). EBC pH did not change during FOT, but FeNO was inversely correlated to EBC pH after the exercise (Spearman's rho=−0.66, P=0.013). With respect to the thermal and physical strain, FOT is considered to be a safe training procedure for healthy firefighters. The increase rate in primary DNA damage found in the instructors' peripheral leukocytes requires further examination in a larger sample size.

The zinc-finger domains of PARP1 cooperate to recognize DNA strand breaks.

Poly(ADP-ribose) polymerase 1 (PARP1) is a primary DNA damage sensor whose (ADP-ribose) polymerase activity is acutely regulated by interaction with DNA breaks. Upon activation at sites of DNA damage, PARP1 modifies itself and other proteins by covalent addition of long, branched polymers of ADP-ribose, which in turn recruit downstream DNA repair and chromatin remodeling factors. PARP1 recognizes DNA damage through its N-terminal DNA-binding domain (DBD), which consists of a tandem repeat of an unusual zinc-finger (ZnF) domain. We have determined the crystal structure of the human PARP1-DBD bound to a DNA break. Along with functional analysis of PARP1 recruitment to sites of DNA damage in vivo, the structure reveals a dimeric assembly whereby ZnF1 and ZnF2 domains from separate PARP1 molecules form a strand-break recognition module that helps activate PARP1 by facilitating its dimerization and consequent trans-automodification.

Mechanisms of radioresistance in terminally differentiated cells of mature retina

Retinopathy of animals is induced by many agents damaging DNA. This fact shows that DNA lesions may initiate retinal degeneration. The aim of our work was to study the effects of gamma and proton irradiation, and methylnitrosourea (MNU) on mice retina. We evaluated morphological changes, DNA damage and repair in retina, and expression of 5 proteins participating in apoptosis: p53, ATM, FasR, PARP and caspase 3 active. Dose of 14 Gy is equitoxic in terms of induction of DNA single strand breaks by both gamma and proton irradiation. But protons were 2 fold more effective than gamma-rays in induction of DNA double strand breaks. All breaks were repaired within < or =10 h. Irradiation resulted in increased expression of p53 and ATM. But no sings of cell death and retinal degeneration were observed during 7 days after irradiation. Proton irradiation in dose of 25 Gy resulted in increasing over time destructive changes localized mainly in photoreceptor layer of retina. These changes were followed by increased expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, caspase 3 active, which was followed by destructive changes in retina with sings of apoptosis of photoreceptors. As in the case of irradiation, the 2-fold dose reduction of MNU abrogated cytotoxic effect of MNU on retina. High level of spontaneous DNA damage such as apurine and apyrimidine sites were observed in mouse retina. The results of our study demonstrate the occurrence of genotoxic threshold in the initiation of retinal cell death in vivo. Topoisomerase 2 of retina is suggested to translate primary DNA damage to cytotoxic effect.

Protective effect of ellagic acid (EA) on micronucleus formation induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in mammalian cells, in in vitro assays and in vivo

The beneficial effects of fruits and vegetables with respect to age-related diseases such as diabetes, atherosclerosis and several types of cancer are widely recognized and confirmed by several epidemiological studies.A possible approach for evaluating the protective potential of promising diet constituents is to evaluate their beneficial effect with respect to a set of biomarkers that are indicative of a potential risk for developing degenerative diseases. Among the numerous biomarkers of the effect of food-related carcinogens and for the assessment of the degree of risk for disease, chromosomal damage detection is very predictive.The aim of this study was to test antigenotoxic effect of ellagic acid (EA) both in in vitro and in vivo studies, in combination with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a methylating agent. EA, a naturally occurring and widely distributed plant phenol, has been intensively studied but with conflicting results, depending on the endpoints considered and the experimental material employed. In vitro and in vivo studies differ in their experimental schedule: in the in vitro study pre- and post-treatments and simultaneous treatments with EA were performed, while in the in vivo study only pre-treatment was carried out.The results of this study clearly demonstrate a protective action of EA with respect to MNNG-induced micronuclei and cell proliferation both in vitro and in vivo. The lack of effect in the post-treatment in in vitro experiments excludes a possible effect of EA on DNA-repair systems. On the other hand, consumption of EA can have a protective action against primary DNA damage induced by oxidative stress.

The use of FISH-comet to detect c-Myc and TP 53 damage in extended-term lymphocyte cultures treated with terbuthylazine and carbofuran

Terbuthylazine and carbofuran are suspected to cause non-Hodgkin's lymphoma and lung cancer. We evaluated the effects of prolonged exposure to low concentrations on primary DNA damage by comet assay, and on the structural integrity of c-Myc and TP 53 genes by FISH-comet. Another novelty in studying these pesticides’ genotoxicity is the use of 14-day extended-term human lymphocyte cultures. Concentrations corresponded to values of ADI and OEL: for terbuthylazine 0.58ng/ml and 8ng/ml; for carbofuran 8ng/ml and 21.6ng/ml, respectively. A possible effect of metabolic activation (S9) was also considered. Carbofuran treatment induced a significant migration of DNA into the tail in a concentration-dependent manner, while for terbuthylazine the effect was significant only at the higher concentration. Terbuthylazine caused migration of both c-Myc signals into the comet tail. A significant occurrence of TP 53 signals in the tail was observed at 8ng/ml. Prolonged carbofuran treatment significantly elevated the migration of a single c-Myc signal into the tail in a concentration-dependent manner. With S9, distribution of signals shifted toward increased presence of both signals in tail. Our results showed impaired structural integrity of c-Myc and TP 53 due to prolonged exposure to terbuthylazine and carbofuran.

In vitro genotoxicity of neutral red after photo-activation and metabolic activation in the Ames test, the micronucleus test and the comet assay

Neutral red (Nr) is relatively non-toxic and is widely used as indicator dye in many biological test systems. It absorbs visible light and is known to act as a photosensitizer, involving the generation of reactive oxygen species (type-I reaction) and singlet oxygen (type-II reaction).The mutagenicity of Nr was determined in the Ames test (with Salmonella typhimurium strains TA1535, TA97, TA98, TA98NR, TA100, and TA102) with and without metabolic activation, and with and without photo-activation on agar plates. Similarly to the situation following metabolic activation, photo-mutagenicity of Nr was seen with all Salmonella strains tested, albeit with different effects between these strains. To our knowledge, Nr is the only photo-mutagen showing such a broad action. Since the effects are also observed in strains not known to be responsive to ROS, this indicates that ROS production is not the sole mode of action that leads to photo-genotoxicity. The reactive species produced by irradiation are short-lived as pre-irradiation of an Nr solution did not produce mutagenic effects when added to the bacteria. In addition, mutagenicity in TA98 following irradiation was stronger than in the nitroreductase-deficient strain TA98NR, indicating that nitro derivatives that are transformed by bacterial nitroreductase to hydroxylamines appear to play a role in the photo-mutagenicity of Nr. Photo-genotoxicity of Nr was further investigated in the comet assay and micronucleus test in L5178Y cells. Concentration-dependent increases in primary DNA damage and in the frequency of micronuclei were observed after irradiation.

Adaptive response in mice exposed to 900 MHz radiofrequency fields: primary DNA damage.

The phenomenon of adaptive response (AR) in animal and human cells exposed to ionizing radiation is well documented in scientific literature. We have examined whether such AR could be induced in mice exposed to non-ionizing radiofrequency fields (RF) used for wireless communications. Mice were pre-exposed to 900 MHz RF at 120 µW/cm2 power density for 4 hours/day for 1, 3, 5, 7 and 14 days and then subjected to an acute dose of 3 Gy γ-radiation. The primary DNA damage in the form of alkali labile base damage and single strand breaks in the DNA of peripheral blood leukocytes was determined using the alkaline comet assay. The results indicated that the extent of damage in mice which were pre-exposed to RF for 1 day and then subjected to γ-radiation was similar and not significantly different from those exposed to γ-radiation alone. However, mice which were pre-exposed to RF for 3, 5, 7 and 14 days showed progressively decreased damage and was significantly different from those exposed to γ-radiation alone. Thus, the data indicated that RF pre-exposure is capable of inducing AR and suggested that the pre-exposure for more than 4 hours for 1 day is necessary to elicit such AR.

Usefulness of combined in vivo skin comet assay and in vivo skin micronucleus test

We have already found that the in vivo skin comet assay is useful for the evaluation of primary DNA damage induced by genotoxic chemicals in epidermal skin cells. The aim of the present study was to evaluate the sensitivity and specificity of the combined in vivo skin comet assay and in vivo skin micronucleus (MN) test using the same animal to explore the usefulness of the new test method.The combined alkaline comet assay and MN test was carried out with three chemicals: 4-nitroquinoline-1-oxide (4NQO), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and benzo[a]pyrene (B[a]P). In the first experiment, we compared DNA- and chromosome-damaging effects of 3 [72, 24 and 3hours (h) before sacrifice] and 4 applications (72, 48, 24 and 3h before sacrifice) of 4NQO, which induces dermal irritancy. The animals were euthanized and their skin was sampled for the combination test. As a result, the 4-application method was able to detect both DNA- and chromosome-damaging potential with a lower concentration; therefore, in the second experiment, MNNG and B[a]P were topically applied four times, respectively. The animals were euthanized, and then their skins were sampled for combination tests. In the alkaline comet assay, significant differences in the percent of DNA (%DNA) in the tail were observed in epidermal skin cells treated with MNNG and B[a]P. In the MN test, an increased frequency of MN cells (%MN) cells was observed by treatment with MNNG; however, there were no significant increases. In contrast, significant differences in %MN were observed by treatment with B[a]P.From these results, we conclude that the combined in vivo skin comet assay and in vivo MN test was useful because it can detect different genotoxicity with the same sampling time and reduce the number of animals used.

Phytochemical, Antioxidant and Toxicological Investigation ofMorinda citrifoliaL. Blossoms

Noni blossoms have a long history of medicinal uses in tropical areas. This study was conducted to investigate the major phytochemical components, toxicological properties, and antioxidant activity of noni blossoms. An HPLC-PDA method was developed and validated for the identification and quantification of major components. The major phytochemicals were iridoid glycosides, deacetylasperulosidic acid and asperulosidic acid, and flavonoids, quercetin-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside and kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside, each present at 3.764, 3.576, 1.513, and 3.096 mg/g, respectively. The aqueous extract of noni blossoms, at 500 μg/mL, exhibited greater antioxidant activity in the 2,2-diphenylpicrylhydrazyl radical scavenging assay than green tea (88.11 ± 0.01% versus 76.60 ± 0.05%). A primary DNA damage test inE. coliPQ37 (SOS-chromotest) and a twenty-four hour brine shrimp toxicity test did not reveal any genotoxic or cytotoxic activity. These results provide a useful reference for the identification of noni blossoms as well as preliminary evaluation of safety and efficacy. Further evaluation of the potential applications of noni blossoms is warranted.