Adapting the in vitro micronucleus assay (OECD Test Guideline No. 487) for testing of manufactured nanomaterials: recommendations for best practices.

The current Organisation for Economic Co-Operation and Development test guideline number 487 (OECD TG No. 487) provides instruction on how to conduct the in vitro micronucleus assay. This assay is one of the gold standard approaches for measuring the mutagenicity of test items; however, it is directed at testing low molecular weight molecules and may not be appropriate for particulate materials (e.g. engineered nanoparticles [ENPs]). This study aimed to adapt the in vitro micronucleus assay for ENP testing and underpins the development of an OECD guidance document. A harmonized, nano-specific protocol was generated and evaluated by two independent laboratories. Cell lines utilized were human lymphoblastoid (TK6) cells, human liver hepatocytes (HepG2) cells, Chinese hamster lung fibroblast (V79) cells, whole blood, and buffy coat cells from healthy human volunteers. These cells were exposed to reference ENPs from the Joint Research Council (JRC): SiO2 (RLS-0102), Au5nm and Au30nm (RLS-03, RLS-010), CeO2 (NM212), and BaSO4 (NM220). Tungsten carbide-cobalt (WC/Co) was used as a trial particulate positive control. The chemical controls were positive in all cell cultures, but WC/Co was only positive in TK6 and buffy coat cells. In TK6 cells, mutagenicity was observed for SiO2- and both Au types. In HepG2 cells, Au5nm and SiO2 showed sub-two-fold increases in micronuclei. In V79 cells, whole blood, and buffy coat cells, no genotoxicity was detected with the test materials. The data confirmed that ENPs could be tested with the harmonized protocol, additionally, concordant data were observed across the two laboratories with V79 cells. WC/Co may be a suitable particulate positive control in the in vitro micronucleus assay when using TK6 and buffy coat cells. Detailed recommendations are therefore provided to adapt OECD TG No. 487 for testing ENP.

Assessment of the three-test genetic toxicology battery for groundwater metabolites.

The two-test in vitro battery for genotoxicity testing (Ames and micronucleus) has in the majority of cases replaced the three-test battery (as two-test plus mammalian cell gene mutation assay) for the routine testing of chemicals, pharmaceuticals, cosmetics, and agrochemical metabolites originating from food and feed as well as from water treatment. The guidance for testing agrochemical groundwater metabolites, however, still relies on the three-test battery. Data collated in this study from 18 plant protection and related materials highlights the disparity between the often negative Ames and in vitro chromosome aberration data and frequently positive in vitro mammalian cell gene mutation assays. Sixteen of the 18 collated materials with complete datasets were Ames negative, and overall had negative outcomes in in vitro chromosome damage tests (weight of evidence from multiple tests). Mammalian cell gene mutation assays (HPRT and/or mouse lymphoma assay (MLA)) were positive in at least one test for every material with this data. Where both MLA and HPRT tests were performed on the same material, the HPRT seemed to give fewer positive responses. In vivo follow-up tests included combinations of comet assays, unscheduled DNA synthesis, and transgenic rodent gene mutation assays, all gave negative outcomes. The inclusion of mammalian cell gene mutation assays in a three-test battery for groundwater metabolites is therefore not justified and leads to unnecessary in vivo follow-up testing.

Measurement of chromosomal instability and level of DNA damage in peripheral blood mononuclear cells of endometrial cancer patients.

Endometrial cancer is one of the most common invasive gynecologic malignancies in developed countries. The aim of this study was to evaluate chromosomal instability and level of DNA damage in peripheral blood mononuclear cells (PBMCs) of newly diagnosed endometrial cancer patients in relation to health status (diagnosis), age, histological grade of cancer, residence, smoking, number of pregnancies, miscarriages, and abortions. The analyzed sample consisted of 60 individuals, 30 endometrial cancer patients with an average age of 64.37 ±â€…7.08, and 30 healthy control women with an average age of 60.23 ±â€…11.55. Chromosomal instability was evaluated by the cytokinesis-block micronucleus (CBMN) assay, and the level of DNA damage by the single-cell gel electrophoresis (comet) assay in PBMCs. The average frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) as well as nuclear buds (NBUDs) were significantly higher in cancer patients compared to controls (P < .0005). There was no difference in the nuclear division index (NDI) among the analyzed samples. The comet assay showed that the patients had a significantly increased genetic damage index (GDI) compared with controls (P < .0005). Using linear regression analysis, we found that health status (diagnosis) had the strongest influence on the MN frequency as well as GDI (P < .0005). Our results indicated that there is a high level of genetic damage in both the level of DNA and the level of chromosomes in the PBMCs of newly diagnosed patients with endometrial cancer, where the frequency and level of damage were significantly affected by health status, grade of cancer, residence, number of pregnancies, miscarriages, and abortions.

Evidence on the genotoxic and ecotoxic effects of PFOA, PFOS and their mixture on human lymphocytes and bacteria.

Considering that the PFOA and PFOS are widely spread chemicals with harmful effects in human and environmental health as well as the increasing interest of the scientific community in the implications that might present especially when they co-exist, this study aims to assess their harmful impacts, both individually and as a mixture on human lymphocytes and aquatic microorganisms. The cytokinesis-block micronucleus (CBMN) assay was used to examine their potential for cytotoxicity and genotoxicity towards human cells, and Microtox assay using Aliivibrio fischeri assay was used to estimate the environmental risk. Regarding the human lymphocytes, the tested concentrations ranged between 250 and 1000 µg L-1, for all cases. PFOA increased slightly the frequency of micronuclei (MN) but without statistical significance. In the case of PFOS, our results showed a dose-dependent increase in the frequency of micronuclei which showed a statistically significant difference (p < 0.001) at 1000 µg L-1, which is the highest studied concentration. Regarding the CBPI index, statistically significant (p < 0.05, p < 0.01, and p < 0.001 respectively) differences were observed at all studied concentrations of PFOS, compared to the control. The mixture was found to be more cytotoxic and genotoxic than the individual tested compounds, causing a higher decrease at the CBPI index even in lower concentrations and increase at the MN frequencies. Aliivibrio fischeri was exposed to various concentrations in the range of 0.5 µg L-1- 20 mg L-1, for 5 and 15 min and significant increase in the inhibition percentage at the highest tested concentration of their mixture after 15 min was observed.

Mobile phone specific radiation disturbs cytokinesis and causes cell death but not acute chromosomal damage in buccal cells: Results of a controlled human intervention study.

Several human studies indicate that mobile phone specific electromagnetic fields may cause cancer in humans but the underlying molecular mechanisms are currently not known. Studies concerning chromosomal damage (which is causally related to cancer induction) are controversial and those addressing this issue in mobile phone users are based on the use of questionnaires to assess the exposure. We realized the first human intervention trial in which chromosomal damage and acute toxic effects were studied under controlled conditions. The participants were exposed via headsets at one randomly assigned side of the head to low and high doses of a UMTS signal (n = 20, to 0.1 W/kg and n = 21 to 1.6 W/kg Specific Absorption Rate) for 2 h on 5 consecutive days. Before and three weeks after the exposure, buccal cells were collected from both cheeks and micronuclei (MN, which are formed as a consequence of structural and numerical chromosomal aberrations) and other nuclear anomalies reflecting mitotic disturbance and acute cytotoxic effects were scored. We found no evidence for induction of MN and of nuclear buds which are caused by gene amplifications, but a significant increase of binucleated cells which are formed as a consequence of disturbed cell divisions, and of karyolitic cells, which are indicative for cell death. No such effects were seen in cells from the less exposed side. Our findings indicate that mobile phone specific high frequency electromagnetic fields do not cause acute chromosomal damage in oral mucosa cells under the present experimental conditions. However, we found clear evidence for disturbance of the cell cycle and cytotoxicity. These effects may play a causal role in the induction of adverse long term health effects in humans.

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing.

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

Genotoxicity and safety pharmacology of the rVSVInd(GML)-mspSGtc vaccine against SARS-CoV-2 in Sprague-Dawley rats and Beagle dogs.

The emergence of coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a pandemic, prompting rapid vaccine development. Although vaccines are effective, the occurrence of rare adverse events following vaccination highlights the necessity of determining whether the benefits outweigh the risks posed by the infection itself. The recombinant Vesicular Stomatitis Virus (rVSV) platform is a promising vector for vaccines against emerging viruses. However, limited studies have evaluated the genotoxicity and safety pharmacology of this viral vector vaccine, which is crucial to ensure the safety of vaccines developed using this platform. Hence, the present study aimed to assess the genotoxicity and safety pharmacology of the rVSVInd(GML)-mspSGtc COVID-19 vaccine using micronucleus and comet assays, as well as neurobehavioral, body temperature, respiratory, and cardiovascular assessments in Sprague-Dawley rats and beagle dogs. The intramuscular administration of rVSVInd(GML)-mspSGtc at doses up to 1.5 × 109 PFU/animal did not increase the number of bone marrow micronucleated polychromatic erythrocytes or cause liver DNA damage. Additionally, it had no significant impact on neurobehavioral functions in rats and showed marginal temporary changes in body temperature, respiratory rate, heart rate, and electrocardiogram parameters in rats and dogs, all of which resolved within 24 h. Overall, following genotoxicity and pharmacological safety assessments, rVSVInd(GML)-mspSGtc displayed no notable systemic adverse effects in rats and dogs, suggesting its potential as a vaccine candidate for human clinical trials.

Study of the DNA damage and cell death in human peripheral blood mononuclear and HepG2/C3A cells exposed to the synthetic 3-(3-hydroxyphenyl)-7-hydroxycoumarin.

Hydroxycoumarins are an important source of biologically active compounds. Previous studies have shown that the number and position of the hydroxyl substituents in the scaffold play an important role for the observed biological activity. In the present study, 3-(3-hydroxyphenyl)-7-hydroxycoumarin was synthesized, and potential cytogenotoxic effects determined in human HepG2/C3A cells displaying phase 1 and phase 2 enzymes (metabolizing cell ability) and compared to human peripheral blood mononuclear cells (PBMC) without xenobiotics metabolizing capacity. Cell viability was determined with concentrations between 0.01 and 10 µg/ml of 3-(3-hydroxyphenyl)-7-hydroxycoumarin using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and trypan blue tests. Genotoxicity was determined utilizing the comet assay, and the clastogenic/aneugenic potential employing the micronucleus (MN) test. The results of the in vitro cytotoxicity assays showed a significant decrease in cell viability of PBMC following exposure to 10 µg/ml concentration of the studied compound after 48 and 72 hr. Comet assay observations noted significant DNA damage in PBMC after 4 hr treatment. No marked cytogenotoxic effects were found in HepG2/C3A cells. No chromosomal mutations were observed in both cell lines. It is important to note that 3-(3-hydroxyphenyl)-7-hydroxycoumarin may exert beneficial pharmacological actions at the low micromolar range and with half-life less than 24 hr. Therefore, the results obtained encourage the continuation of studies on this new molecule for medicinal purposes, but its potential toxicity at higher concentrations and longer exposure times needs to be investigated in further studies.

Presence of micronuclei and nuclear abnormalities in Caracara (Polyborus) plancus living in an airport area in southern Brazil.

The aviation sector is believed to be responsible for considerable environmental damage attributed to emission of a large number and amount of pollutants. Airports are often surrounded by forest fragments and humid areas that attract birds of prey and hence may potentially serve as useful bioindicators. The aim of the present study was to examine genotoxic potential in raptors exposed to airport pollution using the micronucleus (MN) test and morphological changes as evidenced by bilateral symmetry. This investigation was conducted at Salgado Filho International Airport of Porto Alegre - RS as well as in private and zoological breeding grounds. The presence of metals was measured in the blood cells of the collected birds. Seventeen birds (Caracara (Polyborus) plancus) were used in this study 11 from exposed and 6 from non-exposed group. The nuclear alterations clearly indicate that organisms exposed to airport pollution exhibited a significantly higher frequency of genetic damage compared to non-exposed birds. Further, manganese and chromium were detected exclusively in the blood of the exposed group. In contrast, the analysis of bilateral symmetry did not detect any significant morphologic differences between the two groups. Therefore, data indicate that blood genotoxic stress occurs in birds of prey living in civil aviation areas as evidenced by MN frequency increase and presence of manganese and chromium.

Evaluation of infliximab-induced genotoxicity and possible action on BCL-2 and P53 genes.

Although the last pandemic created an urgency for development of vaccines, there was a continuous and concerted effort to search for therapeutic medications among existing drugs with different indications. One of the medications of interest that underwent this change was infliximab (IFM). This drug is used as an anti-inflammatory, predominantly in patients with Crohn 's disease, colitis ulcerative, and rheumatoid arthritis. In addition to these patients, individuals infected with Coronavirus Disease (COVID-19) were administered this chimeric monoclonal antibody (IMF) to act as an immunomodulator for patients in the absence of comprehensive research. Consequently, the present study aimed to examine the genotoxic effects attributed to IFM treatment employing different assays in vivo using mouse Mus musculus. Therefore, IFM was found to induce genotoxic effects as evidenced by the comet assay but did not demonstrate genotoxic potential utilizing mouse bone marrow MN test. The results of evaluating the expression of the P53 and BCL-2 genes using RT-qPCR showed stimulation of expression of these genes at 24 hr followed by a decline at 48 hr. Although the comet assay provided positive results, it is noteworthy that based upon negative findings in the micronucleus test, the data did not demonstrate significant changes in the genetic material that might affect the therapeutic use of IFM. The stimulation of expression of P53 and BCL-2 genes at 24 hr followed by a decline at 48 hr suggest a transient, if any, effect on genetic material. However, there is still a need for more research to more comprehensively understand the genotoxic profile of this medication.

Phytochemical profile and determination of cytotoxicity, acute oral toxicity, genotoxicity, and mutagenicity of aqueous and ethanolic extracts of Pseudobombax marginatum (A. St.-Hil.) A. Robyns.

Pseudobombax marginatum, popularly known as "embiratanha," is widely used by traditional communities as anti-inflammatory and analgesic agent. This study aimed to determine the phytochemical profile as well as cytotoxicity, acute oral toxicity, genotoxicity, and mutagenicity attributed to exposure to aqueous (AqEx) and ethanolic (EtEx) extracts of embiratanha bark. Phytochemical screening was conducted using thin-layer chromatography (TLC). Cell viability was analyzed using MTT assay with human mammary gland adenocarcinoma (MDA-MB-231) and macrophage (J774A.1) cell lines, exposed to concentrations of 12.5, 25, 50, or 100 µg/ml of either extract. For acute oral toxicity, comet assay and micronucleus (MN) tests, a single dose of 2,000 mg/kg of either extract was administered orally to Wistar rats. TLC analysis identified classes of metabolites in the extracts, including cinnamic acid derivatives, flavonoids, hydrolyzable tannins, condensed tannins, coumarins, and terpenes/steroids. In the cytotoxicity assay, the varying concentrations of extracts derived from embiratanha induced no significant alterations in the viability of MDA-MB-231 cells. The lowest concentration of EtEx significantly increased macrophage J774A.1 viability. However, the higher concentrations of AqEx markedly lowered macrophage J774A.1 viability. Animals exhibited no toxicity in the parameters analyzed in acute oral toxicity, comet assay, and MN tests. Further, EtEx promoted a significant reduction in DNA damage index and DNA damage frequency utilizing the comet assay, while the group treated with AqEx exhibited no marked differences. Thus, data demonstrated that AqEx or EtEx of embiratanha may be considered safe at a dose of 2,000 mg/kg orgally under our experimental conditions tested.

Determination of genoprotection against cyclophosphamide induced toxicity in bone marrow of Swiss albino mice by Moringa oleifera leaves and Tinospora cordifolia stem.

The present study aimed to determine the genoprotective activity and safety of Moringa oleifera leave and Tinospora cordifolia stem extracts against cyclophosphamide (CP)-induced genotoxicity utilizing Swiss albino mice. Animals were divided into 14 groups for subacute treatment with either M. oleifera or T. cordifolia extracts daily for 28 days. The extract doses selected were 100, 200 or 400 mg/kg b.w administered orally alone or combined with CP (50 mg/kg b.w. intraperitoneally daily for 5 days). Analyses performed included the comet assay, micronucleus test (MN) in bone marrow cells and sperm head abnormality assay (SHA). M. oleifera and T. cordifolia extracts induced no significant genotoxic effects on somatic and germ cells. In contrast, for all cells examined M. oleifera and T. cordifolia extracts inhibited DNA damage initiated by CP. Taken together data demonstrated that both plant extracts did not exhibit marked genotoxic effects but displayed potential chemoprotective properties against CP-induced genotoxicity in Swiss mice.

Genotoxicity evaluation of food additive titanium dioxide using a battery of standard in vivo tests.

The increasing use of titanium dioxide (TiO2) nanoparticles (NPs) has raised concern about the safety of food additive TiO2. TiO2 has been considered no longer safe by EFSA due to concerns over genotoxicity, however, there are conflicting opinions upon the safety of TiO2 as a food additive, and the number of in vivo genotoxicity studies conducted on food additive TiO2 was limited. In order to investigate the potential genotoxicity of food additive TiO2, we evaluated the genotoxicity of a commercial food additive TiO2 (average size of 135.54 ± 41.01 nm, range from 60.83 to 230.16 nm, NPs account for 30% by number) using a battery of standard in vivo tests, including mammalian erythrocyte micronucleus test, mammalian bone marrow chromosomal aberration test and in vivo mammalian alkaline comet test. After 15 days of consecutive intragastric administration at doses of 250, 500, and 1000 mg/kgBW, food additive TiO2 neither increased the frequencies of bone marrow micronuclei or chromosomal aberration in mice, nor induced DNA strand breakage in rat liver cells. These results indicate that under the condition of this study, food additive TiO2 does not have genotoxic potential although it contains a fraction of NPs.

A comparative study on chromium-induced micronuclei assessment in the peripheral blood of Hsd:ICR mice.

This study investigated the genotoxic effects of chromium (Cr) in Hsd:ICR mice, considering factors such as oxidative state, apoptosis, exposure pathway, duration, pregnancy, and transplacental exposure. Genotoxicity was assessed using the erythrocytes' micronucleus (MN) assay, while apoptosis was evaluated in nucleated blood cells. The results showed that Cr(III) (CrK(SO4 )2 and CrCl3 ) did not induce any marked genotoxic damage. However, Cr(VI) (CrO3 , K2 Cr2 O7 , Na2 Cr2 O7 , and K2 CrO4 ) produced varying degrees of genotoxicity, with CrO3 being the most potent. MN frequencies increased following 24-h exposure, with a greater effect in male mice. Administering 20 mg/kg of CrO3 via gavage did not lead to significant effects compared to intraperitoneal administration. Short-term oral treatment with a daily dose of 8.5 mg/kg for 49 days elevated MN levels only on day 14 after treatment. Pregnant female mice exposed to CrO3 on day 15 of pregnancy exhibited reduced genotoxic effects compared to nonpregnant animals. However, significant increases in MN levels were found in their fetuses starting 48 h after exposure. In summary, data indicate the potential genotoxic effects of Cr, with Cr(VI) forms inducing higher genotoxicity than Cr(III). These findings indicate that gender, exposure route, and pregnancy status might influence genotoxic responses to Cr.

Genotoxic mode of action and threshold exploration of 2-methyl furan under 120-day sub-chronic exposure in male Sprague-Dawley rats.

2-Methylfuran (2-MF) is an important member of the furan family generated during food thermal processing. An in-vivo multiple endpoint genotoxicity assessment system was applied to explore the genotoxic mode of action and threshold of 2-MF. Male Sprague-Dawley rats received 2-MF by oral gavage at doses of 0.16, 0.625, 2.5, and 10 mg/kg.bw/day for 120 days. An additional 15 days were granted for recovery. The Pig-a gene mutation frequency of RET and RBC showed significant increases among the 2-MF groups on day 120. After a 15-day recovery period, the Pig-a gene mutation frequency returned to levels similar to those in the vehicle control. The tail intensity (TI) values of peripheral blood cells at a dose of 10 mg/kg.bw/day significantly increased from day 4 and remained at a high level after the recovery period. No statistical difference was found in the micronucleus frequency of peripheral blood between any 2-MF dose group and the corn oil group at any timepoint. 2-MF may not induce the production of micronuclei, but it could cause DNA breakage. It could not be ruled out that 2-MF may accumulate in vivo and cause gene mutations. Hence, DNA, other than the spindle, may be directly targeted. The mode of action of 2-MF may be that it was metabolized by EPHX1 to more DNA-active metabolites, thus leading to oxidative and direct DNA damage. The point of departure (PoD) of 2-MF-induced genotoxicity was derived as 0.506 mg/kg bw/day.

Assessment of imidacloprid induced genotoxicity in Pethia conchonius (Rosy barb), a common freshwater fish of India.

Imidacloprid is one of the highly efficient, globally used neonicotinoid groups of insecticides. The indiscriminate use of imidacloprid is contaminating large water bodies affecting not only the target organisms but also non-target organisms including fish. The present study aimed to assess the extent of nuclear DNA damage by imidacloprid in Pethia conchonius a freshwater fish in India using comet and micronucleus assays. The LC50 value of imidacloprid was estimated to be 227.33 mg L-1. Based on the LC50-96 h value, three sub-lethal concentrations of imidacloprid, SLC I -18.94 mg L-1, SLC II -28.41 mg L-1 and SLC III -56.83 mg L-1 were used to detect its genotoxic effect at DNA and cellular level. The imidacloprid exposed fishes exhibited higher DNA damage and nuclear abnormalities (p < 0.05) than the control. The %head DNA, %tail DNA, tail length and the frequency of micronuclei with other nuclear abnormalities like blebbed and notched nuclei were significantly higher than the control in a time and concentration-dependent manner. The DNA damage parameters such as %head DNA (29.107 ± 1.843), %tail DNA (70.893 ± 1.843), tail length (361.431 ± 8.455) micronucleus (1.300 ± 0.019), notched (0.844 ± 0.011) and blebbed (0.811 ± 0.011) nuclei were found to be highest for SLC III (56.83 mg L-1) at 96 h. The findings indicate that IMI is highly genotoxic in fish and other vertebrates leading to mutagenic/clastogenic effects. The study will be helpful in optimization of the imidacloprid use.

In vitro evaluation of the selective cytotoxicity and genotoxicity of three synthetic ortho-nitrobenzyl derivatives in human cancer cell lines, with and without metabolic activation.

Although the presence of nitro groups in chemicals can be recognized as structural alerts for mutagenicity and carcinogenicity, nitroaromatic compounds have attracted considerable interest as a class of agents that can serve as source of potential new anticancer agents. In the present study, the in vitro cytotoxicity, genotoxicity, and mutagenicity of three synthetic ortho-nitrobenzyl derivatives (named ON-1, ON-2 and ON-3) were evaluated by employing human breast and ovarian cancer cell lines. A series of biological assays was carried out with and without metabolic activation. Complementarily, computational predictions of the pharmacokinetic properties and druglikeness of the compounds were performed in the Swiss ADME platform. The MTT assay showed that the compounds selectively affected selectively the cell viability of cancer cells in comparison with a nontumoral cell line. Additionally, the metabolic activation enhanced cytotoxicity, and the compounds affected cell survival, as demonstrated by the clonogenic assay. The comet assay, the cytokinesis-block micronucleus assay, and the immunofluorescence of the γ-H2AX foci formation assay have that the compounds caused chromosomal damage to the cancer cells, with and without metabolic activation. The results obtained in the present study showed that the compounds assessed were genotoxic and mutagenic, inducing double-strand breaks in the DNA structure. The high selectivity indices observed for the compounds ON-2 and ON-3, especially after metabolic activation with the S9 fraction, must be highlighted. These experimental biological results, as well as the theoretical properties predicted for the compounds have shown that they are promising anticancer candidates to be exploited in additional studies.

Assessment of genotoxicity biomarkers in gasoline station attendants due to occupational exposure.

Gasoline station attendants are exposed to numerous chemicals that might have genotoxic and carcinogenic potential, such as benzene in fuel vapor and particulate matter and polycyclic aromatic hydrocarbons in vehicle exhaust emission. According to IARC, benzene and diesel particulates are Group 1 human carcinogens, and gasoline has been classified as Group 2A "possibly carcinogenic to humans." At gas stations, self-service is not implemented in Turkey; fuel-filling service is provided entirely by employees, and therefore they are exposed to those chemicals in the workplace during all working hours. Genetic monitoring of workers with occupational exposure to possible genotoxic agents allows early detection of cancer. We aimed to investigate the genotoxic damage due to exposures in gasoline station attendants in Turkey. Genotoxicity was evaluated by the Comet, chromosomal aberration, and cytokinesis-block micronucleus assays in peripheral blood lymphocytes. Gasoline station attendants (n = 53) had higher tail length, tail intensity, and tail moment values than controls (n = 61). In gasoline station attendants (n = 46), the frequencies of chromatid gaps, chromosome gaps, and total aberrations were higher compared with controls (n = 59). Increased frequencies of micronuclei and nucleoplasmic bridges were determined in gasoline station attendants (n = 47) compared with controls (n = 40). Factors such as age, duration of working, and smoking did not have any significant impact on genotoxic endpoints. Only exposure increased genotoxic damage in gasoline station attendants independently from demographic and clinical characteristics. Occupational exposure-related genotoxicity risk may increase in gasoline station attendants who are chronically exposed to gasoline and various chemicals in vehicle exhaust emissions.

Is micronucleus assay in nasal mucosa cells an appropriate technique for detecting genotoxins by inhalation in humans? A systematic review.

This study aimed to evaluate the scientific literature on the micronucleus assay in nasal mucosa as an appropriate method for evaluating genotoxicity caused by chemical agents. According to the PRISMA guidelines, only in vivo human studies with micronucleus assays using nasal cells were considered. Reviews, case reports, editorials, letters to the editor, and articles not written in English were excluded. The following scientific databases/search engines were used: PubMed/MEDLINE, Scopus, and Web of Science. Results: This review included 13 studies. Four articles detected no statistical significance regarding the frequency of micronuclei while nine articles showed an increase in micronuclei in nasal cells. In the qualitative analysis, two articles were considered strong, eight were moderate and three were weak. The micronucleus assay using nasal mucosa cells is a sensitive and effective technique for assessing DNA damage and an appropriate method for monitoring humans continuously exposed to chemicals.

Subjective biosafety assessment of bisdemethoxycurcumin from the rhizomes of Curcuma longa.

Introduction/Background: Curcuma longa, a plant native to the Indian subcontinent has a variety of biological activities. Curcumin is the most abundant and biologically active compound with many therapeutic properties. Demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) - the two other bioactive components present in Curcuma longa, besides curcumin, are collectively termed curcuminoids. Apart from the well-known curcumin, BDMC also has been reported to possess promising biological and pharmacological effects, but very little scientific evidence on its safety assessment has been published.Objective: The present study was undertaken to determine the safety of pure BDMC from Curcuma longa extract in rodents which comprises of general toxicity (both four weeks and three months duration), reproductive/developmental toxicity and genotoxicity studies.Methods: The Good Laboratory Practice studies were carried out in accordance with the test guidelines established by the Organization for Economic Cooperation and Development.Results: No treatment-related adverse findings were seen in general toxicity testing and a no observed adverse effect level (NOAEL) of 1000 mg/kg/day was established after four weeks (sub-acute) and three-months (sub-chronic) dosing. Evaluation of fertility, embryo-fetal, and post-natal reproductive and developmental parameters also showed no adverse findings with a NOAEL of 1000 mg/kg/day established. The results of genotoxicity as evaluated by in vitro reverse mutation assay, and in vivo micronucleus test in mice indicate that BDMC did not induce any genotoxic effects.Conclusion: Oral administration of BDMC is safe in rodents and non-mutagenic, with no adverse effects under experimental conditions.