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.

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.

Genotoxicological and physiological effects of glyphosate and its metabolite, aminomethylphosphonic acid, on the freshwater invertebrate Lymnaea stagnalis.

Aminomethylphosphonic acid (AMPA) is the main metabolite in the degradation of glyphosate, a broad-spectrum herbicide, and it is more toxic and persistent in the environment than the glyphosate itself. Owing to their extensive use, both chemicals pose a serious risk to aquatic ecosystems. Here, we explored the genotoxicological and physiological effects of glyphosate, AMPA, and the mixed solution in the proportion 1:1 in Lymnaea stagnalis, a freshwater gastropod snail. To do this, adult individuals were exposed to increasing nominal concentrations (0.0125, 0.025, 0.050, 0.100, 0.250, 0.500 µg/mL) in all three treatments once a week for four weeks. The genotoxicological effects were estimated as genomic damage, as defined by the number of micronuclei and nuclear buds observed in hemocytes, while the physiological effects were estimated as the effects on somatic growth and egg production. Exposure to glyphosate, AMPA, and the mixed solution caused genomic damage, as measured in increased frequency of micronuclei and nuclear buds and in adverse effects on somatic growth and egg production. Our findings suggest the need for more research into the harmful and synergistic effects of glyphosate and AMPA and of pesticides and their metabolites in general.

Evaluation of genotoxic effect via expression of DNA damage responsive gene induced by ivermectin on MDBK cell line.

Ivermectin (IVM) is an anti-parasitic drug which is used for treating parasitic infestations. It has been used in humans for treating intestinal strongyloidiasis and onchocerciasis however, currently researchers are investigating its potential for treating coronavirus SARS-CoV-2. Due to its broad-spectrum activities, IVM is being used excessively in animals which has generated an interest for researchers to investigate its toxic effects. Cytotoxic and genotoxic effects have been reported in animals due to excessive usage of IVM. Therefore, this study aims to evaluate the cytotoxic and genotoxic effects of IVM on the Madin-Darby-Bovine-Kidney (MDBK) cell line by examining the expression of a DNA damage-responsive gene (OGG1). Cytotoxicity of IVM was tested using an assay (MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), whereas the genotoxicity was evaluated using comet assay along with micronucleus assay. Moreover, the gene expression of DNA damage response gene (OGG1) was measured by qRT-PCR, after extraction of RNA from the MDBK cell line using the TRIzol method and its conversion to cDNA by reverse-transcriptase PCR. During the experiment, cell viability percentage was measured at different doses of IVM i.e., 25%, 50%, 75%, along with LC50/2, LC50 and LC50*2. It was observed that the gene expression of OGG1 increased as the concentration of IVM increased. It was concluded that IVM has both cytotoxic and genotoxic effects on the MDBK cell line. Furthermore, it is recommended that studies related to the toxic effects of IVM at molecular level and on other model organisms should be conducted to combat its hazardous effects.

A pilot study exploring time- and dose-dependent DNA damage and chromosomal instability caused by benzo[a]pyrene in two urothelial cell types.

Environmental and occupational exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with adverse health effects in humans. Uncertainty exists regarding the causation of urinary bladder cancer by benzo[a]pyrene (B[a]P) due to a lack of sufficient data. In this work, we focused on in-vitro DNA damage and the formation of micronuclei and chromosomal aberrations as predictors of cancer risk, applying a wide range of dosages and time periods to quantify the onset, intensity, and duration of the response. We chose two urothelial cell types to compare susceptibility and the ability to increase the malignity of a pre-existing bladder cancer: a cancer cell line (T24) and a pooled sample of primary urinary bladder epithelia cells (PUBEC) from pigs. The highest level of DNA damage assessed by comet assay was observed following 24-h treatment in both cell types, whereas PUBEC cells were clearly more susceptible. Even 4-h treatment induced DNA damage in PUBEC cells with benchmark doses of 0.0027 µM B[a]P and 0.00023 µM after 4-h and 24-h exposure, respectively. Nearly no effect was observed for periods of 48 h. The frequency of micronucleus formation increased more markedly in T24 cells, particularly with 24-h treatment. In PUBEC cells, 48-h exposure notably induced the formation of nucleoplasmic bridges and nuclear buds. Even though only one biological replicate was studied due to the sophisticated study design, our results give a strong indication of the potential of B[a]P to induce and increase malignity in human-relevant cell types.

In vitro toxicological evaluation of glo menthol and non-menthol heated tobacco products.

Heated tobacco products (HTPs) are non-combustible, inhaled tobacco products that generate an aerosol with fewer and lower levels of toxicants, with a potential to reduce risk relative to cigarette smoking. Here, we assessed in vitro toxicological effects of three menthol (glo neo neoCLICK, neo Smooth Menthol and Fresh Menthol) and one non-menthol (neo Smooth Tobacco) variants of glo HTP, along with market comparators for cigarettes and HTPs. Limited chemical characterization of the study products revealed significantly lower levels of acetaldehyde, acrolein, crotanaldehyde and formaldehyde in test samples from HTPs than those from cigarettes. The glo HTPs were non-mutagenic in the bacterial reverse mutagenesis assay. Although, the whole aerosol exposures of glo HTPs were classified as genotoxic in the in vitro micronucleus assay, and cytotoxic in the NRU (monolayer) and MTT (3 dimensional EpiAirway™ tissues) assays, the cigarette comparators were the most toxic study products in each of these assessments. Further, glo HTPs elicited oxidative stress responses only at the highest dose tested, whereas the cigarette comparators were potent inducers of oxidative stress at substantially lower doses in the EpiAirway tissues. The comparator (non-glo) HTP results were similar to the glo HTPs in these assays. Thus, the glo HTPs exhibit substantially lower toxicity compared to cigarettes.

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.

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.

Exploring cytokinesis block micronucleus assay in Croatia: A journey through the past, present, and future in biomonitoring of the general population.

In this study, we used the cytokinesis-block micronucleus (CBMN) assay to evaluate the background frequency of cytogenetic damage in peripheral blood lymphocytes of the general population concerning different anthropometric data and lifestyle factors. The background frequency of CBMN assay parameters was analysed in 850 healthy, occupationally non-exposed male and female subjects (average age, 38±11 years) gathered from the general Croatian population from 2000 to 2023. The mean background values for micronuclei (MNi) in the whole population were 5.3±4.3 per 1000 binucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 0.7±1.3 and of nuclear buds (NBUDs) 3.1±3.2. The cut-off value, which corresponds to the 95th percentile of the distribution of 850 individual values, was 14 MNi, 3 NPBs, and 9 NBUDs. Results from our database also showed an association of the tested genomic instability parameters with age and sex but also with other lifestyle factors. These findings underscore the importance of considering several anthropometric and lifestyle factors when conducting biomonitoring studies. Overall, the normal and cut-off values attained here present normal values for the general population that can later serve as baseline values for further human biomonitoring studies either in Croatia or worldwide.

The Micronucleus Assay on Cryopreserved Whole Blood.

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, and in vitro radiosensitivity testing. This cytogenetic method is based on the detection of micronuclei in binucleated cells resulting from chromosomal fragments lagging during cell division. Fresh whole blood samples are the most preferred sample type for the CBMN assay. However, the disadvantages of working with fresh blood samples include immediate processing after blood collection and the limited number of repeated analyses that can be performed without extra blood sampling. As the need for fresh blood samples can be logistically challenging, CBMN assay on cryopreserved whole blood samples would be of great advantage, especially in large-scale patient studies. This paper describes a protocol to freeze whole blood samples and to perform the CBMN assay on these frozen blood samples. Blood samples from healthy volunteers have been frozen and thawed at different time points and then, subjected to a modified micronucleus assay protocol. The results demonstrate that this optimized procedure allows the performance of the CBMN assay on frozen blood samples. The described cryopreservation protocol may also be very useful for other cytogenetic assays and a variety of functional assays requiring proliferating lymphocytes.

Metabolism-dependent mutagenicity of two structurally similar tobacco-specific nitrosamines (N-nitrosonornicotine and N-nitrosoanabasine) in human cells, partially different CYPs being activating enzymes.

N-nitrosonornicotine (NNN) and N-nitrosoanabasine (NAB) are both tobacco-specific nitrosamines bearing two heterocyclic amino groups, NAB bearing an extra -CH2- group (conferring a hexa- rather than penta-membered cycle) but with significantly decreased carcinogenicity. However, their activating enzymes and related mutagenicity remain unclear. In this study, the chemical-CYP interaction was analyzed by molecular docking, thus the binding energies and conformations of NNN for human CYP2A6, 2A13, 2B6, 2E1 and 3A4 appeared appropriate as a substrate, so did NAB for human CYP1B1, 2A6, 2A13 and 2E1. The micronucleus test in human hepatoma (HepG2) cells with each compound (62.5-1000 µM) exposing for 48 h (two-cell cycle) was negative, however, pretreatment with bisphenol AF (0.1-100 nM, CYPs inducer) and ethanol (0.2% v:v, CYP2E1 inducer) potentiated micronucleus formation by both compounds, while CITCO (1 µM, CYP2B6 inducer) selectively potentiated that by NNN. In C3A cells (endogenous CYPs enhanced over HepG2) both compounds induced micronucleus, which was abolished by 1-aminobenzotriazole (60 µM, CYPs inhibitor) while unaffected by 8-methoxypsoralen (1 µM, CYP2A inhibitor). Consistently, NNN and NAB induced micronucleus in V79-derived recombinant cell lines expressing human CYP2B6/2E1 and CYP1B1/2E1, respectively, while negative in those expressing other CYPs. By immunofluorescent assay both compounds selectively induced centromere-free micronucleus in C3A cells. In PIG-A assays in HepG2 cells NNN and NAB were weakly positive and simply negative, respectively; however, in C3A cells both compounds significantly induced gene mutations, NNN being slight more potent. Conclusively, both NNN and NAB are mutagenic and clastogenic, depending on metabolic activation by partially different CYP enzymes.

Investigations on the genotoxic potential of styrene in Fischer 344 rats using multiple endpoints.

Genotoxicity of styrene monomer was evaluated in male Fischer 344 rats using the alkaline comet assay for DNA damage, micronucleus assay for cytogenetic damage and the Pig-a assay for gene mutations. In a dose range finding (DRF) study, styrene was administered by oral gavage in corn oil for 28 consecutive days at 0, 100, 500, and 1000 mg/kg/day. The bioavailability of styrene was confirmed in the DRF by measuring its plasma levels at approximately 7- or 15-min following dosing. The 1000 mg/kg/day group exceeded the maximum tolerated dose based on body weight and organ weight changes and signs of central nervous system depression. Based on these findings, doses of 0, 100, 250, and 500 mg/kg/day (for 28 or 29 days) were selected for the genotoxicity assays. Animals were sacrificed 3-4 h after treatment on Day 28 or 29 for assessing various genotoxicity endpoints. Pig-a mutant frequencies and micronucleus frequencies were determined in peripheral blood erythrocytes. The comet assay was conducted in the glandular stomach, duodenum, liver, lung, and kidney. These studies were conducted in accordance with the relevant OECD test guidelines. Oral administration of styrene did not lead to genotoxicity in any of the investigated endpoints. The adequacy of the experimental conditions was assured by including animals treated by oral gavage with the positive control chemicals ethyl nitrosourea and ethyl methane sulfonate. Results from these studies supplement to the growing body of evidence suggesting the lack of in vivo genotoxic potential for styrene.

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.

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.

Safety assessment of high fructose corn syrup and fructose used as sweeteners in foods.

High Fructose Corn Syrup (HFCS) and Fructose (FR) are widely used sweeteners in many foods and beverages. This study aimed at investigating the cytotoxic effects of HFCS (5%-30%) and FR (62.5-2000 µg/mL) using MTT assay in Human Hepatocellular Carcinoma (HepG2) cells, and genotoxic effects of using Chromosome Aberrations (CAs), Sister Chromatid Exchanges (SCEs), Micronuclei (MN) and comet assays in human lymphocytes. HFCS significantly reduced the cell viability in HepG2 cells at between 7.5% and 30% for 24 and 48 h. 30% HFCS caused a very significant toxic effect. FR had a cytotoxic effect in HepG2 cells at all treatments. However, as fructose concentration decreased, the cell viability decreased. HFCS (10%-20%) and FR (250-2000 µg/mL) decreased the mitotic index at higher concentrations. IC50 value was found to be a 15% for 48 h. IC50 value of FR was detected as 62.5 µg/mL for 24 h and 48 h. HFCS significantly increased CAs frequency at 15% and 20%. FR significantly increased the frequency of CAs at 250, 1000, and 2000 µg/mL for 48 h. Both sweeteners increased the frequency of SCEs at all concentrations. HFCS (15% and 20%) and FR (250, 1000, and 2000 µg/mL) induced MN frequency at higher concentrations. HFCS caused DNA damage in comet assay at 10% -30%. FR increased tail intensity and moment at 125-2000 µg/mL and tail length at 62.5, 250 and 500 µg/mL. Therefore, HFCS and FR are clearly seen to be cytotoxic and genotoxic, especially at higher concentrations.

HFCS and FR exhibited cytotoxic effect at HepG2 and human lymphocytes at higher concentrations.Both sweeteners increased the frequencies of CAs and SCEs at higher concentrations.HFCS caused DNA damage at 10% -30% concentrations.HFCS (15% and 20%) and FR (250, 1000, and 2000 µg/mL) induced MN frequency.

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.

Comparative analysis of micronucleus induction and DNA damage biomarkers in TK6 and A375 cells using flow cytometry.

Previously, we introduced an alternative adherent A375 cell line for clastogenicity and aneugenicity testing using a high content imaging platform. To further characterize the performance of A375 cells, we investigated the sensitivity and specificity of A375 and TK6 cells by directly comparing micronucleus (MN) induction, cytotoxicity (relative cell counts, viability, and apoptosis), clastogenicity (γH2AX), and aneuploidy markers (pH 3, MPM-2, and polyploidy) using flow cytometric methods. We evaluated 14 compounds across different mechanisms (non-genotoxic apoptosis inducers, clastogens, and aneugens with either tubulin binding or aurora kinase inhibiting phenotypes) at 4-h and 24-h post treatment. Both aneugens and clastogens tested positive for micronucleus induction in both cell lines. Apoptosis continued to be a confounding factor for flow cytometry-based micronuclei assessment in TK6 cells as evidenced by positive responses by the three cytotoxicants. Conversely, A375 cells were not affected by apoptosis-related false positive signals and did not produce a positive response in the in vitro micronucleus assay. Benchmark dose response (BMD) analysis showed that the induction of micronuclei and biomarkers occurred at similar concentrations in both cell lines for clastogens and aneugens. By showing that A375 cells have similar sensitivity to TK6 cells but a greater specificity, these results provide additional support for A375 cells to be used as an alternative adherent cell line for in vitro genetic toxicology assessment.

Veterinarians exposed to inhaled anesthetic present chromosome damage, apoptosis and cell cycle changes.

This cross-sectional study evaluated, for the first time, DNA damage, viability, and cell death of lymphocytes and cell cycle phases of mononuclear and polymorphonuclear cells in veterinarians exposed to the volatile anesthetic isoflurane. Veterinarians who were occupationally exposed to isoflurane (exposed group; n = 20) and matched-unexposed individuals (volunteers without occupational exposure; n = 20) were enrolled in the study. DNA damage was assessed in lymphocytes by micronucleus (MN) and phosphorylated histone gamma-H2AX (γ-H2AX). Cell viability, cytotoxicity, and the cell cycle were evaluated by flow cytometry. Isoflurane was detected in urine samples by headspace gas chromatography-mass spectrometry. Compared with unexposed subjects, veterinarians occupationally exposed to isoflurane (25.7 ± 23.7 µg/L urine) presented statistically higher MN frequencies, lymphocytic apoptosis rates, and numbers of polymorphonuclear cells in the G0/G1 stage. Additionally, the exposed group presented statistically lower proportions of viable lymphocytes and G2/M polymorphonuclear cells. Our findings indicate that veterinarians who are frequently exposed to inhaled anesthetic exhibit chromosomal and cell damage in addition to changes in peripheral blood cell proliferation.

Cytogenotoxicity effects in addicts with multidrug consumption.

Drug abuse is considered a global health problem with serious social impact. In recent decades, changes in drug consumption patterns have shown a clear rising trend in the use of multiple drugs. Although the buccal micronucleus cytome (BMCyt) assay has evaluated cytotoxicity in drug abuse, there has not been an approach that takes into account this pattern of multiple drug use. Therefore, in this study, we evaluate for the first time the cytogenotoxic effects in multidrug users, and its correlation with the amount consumed and years of abuse. This study was conducted on 166 individuals by the BMCyt assay. A total of 83 individuals with a history of multiple licit (alcohol and tobacco) and at least one illicit drug abuse (marijuana, methamphetamines, cocaine, and/or inhalants), and 83 healthy individuals, non-drug abusers were analyzed. The results showed that drug abusers had higher frequencies of nuclear abnormalities nuclear buds, binucleated cells, pyknotic nuclei (PNs), karyorrhexis (KX), and abnormally condensed chromatin when compared with healthy controls. Moreover, results suggests that the use of licit and illicit drugs is related to cytogenotoxic damage, as was shown by an upward trend in the frequency of nuclear abnormalities identified in groups 1 (alcohol + tobacco + at least one illicit drug) and 2 (tobacco + at least one illicit drug). Furthermore, a positive correlation was found in the different groups, between the years and the amount of consumption of some drugs (alcohol, methamphetamine, and tobacco) with cytotoxicity markers such as KL, KX, and PNs.

A toxicological assessment of spermidine trihydrochloride produced using an engineered strain of Saccharomyces cerevisiae.

Spermidine is a polyamine consumed in the diet, endogenously biosynthesized in most cells, and produced by the intestinal microbiome. A variety of foods contribute to intake of spermidine along with other polyamines. Spermidine trihydrochloride (spermidine-3HCl) of high purity can be produced using an engineered strain of Saccharomyces cerevisiae. Spermidine has a demonstrated history of safe use in the diet; however, limited information is available in the public literature to assess the potential toxicity of spermidine-3HCl. To support a safety assessment for this spermidine-3HCl as a dietary source of spermidine, authoritative guideline and good laboratory practice (GLP) compliant in vitro genotoxicity assays (bacterial reverse mutation and mammalian micronucleus assays) and a 90-day oral (dietary) toxicity study in rats were conducted with spermidine-3HCl. Spermidine-3HCl was non-genotoxic in the in vitro assays, and no adverse effects were reported in the 90-day oral toxicity study up to the highest dose tested, 12500 ppm, equivalent to 728 mg/kg bw/day for males and 829 mg/kg bw/day for females. The subchronic no observed adverse effect level (NOAEL) is 728 mg/kg bw/day.