In vivo micronucleus assay on sodium molybdate in rats and its impact on the overall assessment of the genotoxicity of molybdenum substances.

In this paper we present methodological and experimental details and results from an OECD Test Guideline 474 and GLP-compliant in vivo micronucleus study on sodium molybdate dihydrate in Sprague Dawley rats. Prior to the conduct of this study, there was a data-gap for reliable in vivo genotoxicity data for molybdenum substances. The presentation of the new study is complemented by a review of other available in vitro and in vivo data on the genotoxicity of molybdenum substances, focussing on substances where the contained or released molybdate ion, MoO42-, is considered the responsible moiety for any toxicological effect (grouping/category approach). After consideration of the relevance and reliability of all available data, the absence of a concern for genotoxicity of molybdate in vitro and in vivo is concluded.

Hydromorphone impurity 2,2-bishydromorphone does not exert mutagenic and clastogenic properties via in silico QSAR prediction and in vitro Ames and micronucleus test.

The opioid agonist hydromorphone is indicated for the management of severe acute and chronic pain given that alternate treatments are insufficient. While the genotoxicity profile of hydromorphone is well investigated, little is known about the genotoxic potential of its impurities. In this study, 2,2-bishydromorphone was tested in silico and in vitro for both its mutagenic potential in an Ames test performed with Salmonella typhimurium and Escherichia coli tester strains up to a maximum concentration of 5 mg per plate in the absence and presence of metabolic activation. Furthermore, it was tested for its ability to induce micronuclei in TK6 cells in a micronucleus test up to a maximum concentration of 500 µg/mL with or without an exogenous metabolic activation system. 2,2-Bishydromorphone did not reveal any potential for inducing mutagenicity or clastogenicity under the conditions of the respective tests and is therefore considered non-mutagenic and non-clastogenic/aneugenic in vitro. These results are in line with negative in silico quantitative structure-activity relationship (QSAR) prediction for 2,2-bishydromorphone mutagenicity and clastogenicity and provide evidence of good correlation of in silico and in vitro data. Conclusively, these studies add important new clinically relevant information on the safety of hydromorphone as the impurity of 2,2-bishydromorphone is proven to be non-mutagenic and non-clastogenic.

Absence of hydroxyurea-induced mutational effects supports higher utilisation for the treatment of sickle cell anaemia.

Hydroxyurea (hydroxycarbamide) is approved for treating both children and adults with sickle cell anaemia (SCA). Fetal haemoglobin (HbF) induction is the primary treatment response, along with improved anaemia, reduced haemolysis, myelosuppression and decreased endothelial inflammation. Hydroxyurea has proven clinical efficacy for SCA - treatment significantly reduces disease manifestations and prolongs survival. Despite these recognised benefits, long-standing concerns regarding the risks of mutagenic and potentially carcinogenic drug exposure have hampered efforts for broad hydroxyurea use in SCA, although these are based largely on outdated experimental models and treatment experiences with myeloproliferative neoplasms. Consequently, many patients with SCA are not receiving this highly effective disease-modifying therapy. In this review, we describe the concept of genotoxicity and its laboratory measurements, summarise hydroxyurea-associated data from both preclinical and clinical studies, and discuss carcinogenic potential. The genotoxicity results clearly demonstrate that hydroxyurea does not directly bind DNA and is not mutagenic. Rather, its genotoxic effects are limited to indirect clastogenicity occurring in select cell types, and only when high dose and time thresholds are exceeded. This absence of mutagenic activity is consistent with the observed lack of any compelling carcinogenic potential. Since hydroxyurea therapy for SCA carries minimal carcinogenic risks, the current drug labelling should be modified accordingly, and prescribing practices should be broadened to allow better access and increased utilisation of this highly effective drug.

A comprehensive weight of evidence assessment of published acetaminophen genotoxicity data: Implications for its carcinogenic hazard potential.

In 2019, the California Office of Environmental Health Hazard Assessment initiated a review of the carcinogenic hazard potential of acetaminophen, including an assessment of its genotoxicity. The objective of this analysis was to inform this review process with a weight-of-evidence assessment of more than 65 acetaminophen genetic toxicology studies that are of widely varying quality and conformance to accepted standards and relevance to humans. In these studies, acetaminophen showed no evidence of induction of point or gene mutations in bacterial and mammalian cell systems or in in vivo studies. In reliable, well-controlled test systems, clastogenic effects were only observed in unstable, p53-deficient cell systems or at toxic and/or excessively high concentrations that adversely affect cellular processes (e.g., mitochondrial respiration) and cause cytotoxicity. Across the studies, there was no clear evidence that acetaminophen causes DNA damage in the absence of toxicity. In well-controlled clinical studies, there was no meaningful evidence of chromosomal damage. Based on this weight-of-evidence assessment, acetaminophen overwhelmingly produces negative results (i.e., is not a genotoxic hazard) in reliable, robust high-weight studies. Its mode of action produces cytotoxic effects before it can induce the stable, genetic damage that would be indicative of a genotoxic or carcinogenic hazard.

Induction of chromosomal and DNA damage and histological alterations by graphene oxide nanoparticles in Swiss mice.

The unique physicochemical properties of graphene oxide (GO) nanoparticles increase their uses in a wide range of applications that increase their release into the environment, and thus human exposure. However, the in vivo clastogenicity and genotoxicity of GO nanoparticles have not been well investigated. The current study was, therefore, designed to investigate the possible induction of chromosomal and DNA damage by GO nanoparticles and their impact on the tissue architecture in mice. Oral administration of GO nanoparticles for one or five consecutive days at the three dose levels 10, 20 or 40 mg/kg significantly increased the micronuclei and DNA damage levels in a dose-dependent manner in mice bone marrow cells, as well as caused, histological lesions including apoptosis, necrosis, inflammations and cells degeneration in the mice liver and brain tissue sections compared to the normal control mice. Thus, we concluded that oral administration of GO nanoparticles induced chromosomal and DNA damage in a dose-dependent manner as well as histological injuries in both acute and subacute treatments.

Oxidative Stress, Mutations and Chromosomal Aberrations Induced by In Vitro and In Vivo Exposure to Furan.

Furan is a volatile compound that is formed in foods during thermal processing. It is classified as a possible human carcinogen by international authorities based on sufficient evidence of carcinogenicity from studies in experimental animals. Although a vast number of studies both in vitro and in vivo have been performed to investigate furan genotoxicity, the results are inconsistent, and its carcinogenic mode of action remains to be clarified. Here, we address the mutagenic and clastogenic activity of furan and its prime reactive metabolite cis-2 butene-1,4-dial (BDA) in mammalian cells in culture and in mouse animal models in a search for DNA lesions responsible of these effects. To this aim, Fanconi anemia-derived human cell lines defective in the repair of DNA inter-strand crosslinks (ICLs) and Ogg1-/- mice defective in the removal of 8-hydroxyguanine from DNA, were used. We show that both furan and BDA present a weak (if any) mutagenic activity but are clear inducers of clastogenic damage. ICLs are strongly indicated as key lesions for chromosomal damage whereas oxidized base lesions are unlikely to play a critical role.

Benchmark dose analyses of multiple genetic toxicity endpoints permit robust, cross-tissue comparisons of MutaMouse responses to orally delivered benzo[a]pyrene.

Genetic damage is a key event in tumorigenesis, and chemically induced genotoxic effects are a human health concern. Although genetic toxicity data have historically been interpreted using a qualitative screen-and-bin approach, there is increasing interest in quantitative analysis of genetic toxicity dose-response data. We demonstrate an emerging use of the benchmark dose (BMD)-approach for empirically ranking cross-tissue sensitivity. Using a model environmental carcinogen, we quantitatively examined responses for four genetic damage endpoints over an extended dose range, and conducted cross-tissue sensitivity rankings using BMD100 values and their 90% confidence intervals (CIs). MutaMouse specimens were orally exposed to 11 doses of benzo[a]pyrene. DNA adduct frequency and lacZ mutant frequency (MF) were measured in up to 8 tissues, and Pig-a MF and micronuclei (MN) were assessed in immature (RETs) and mature red blood cells (RBCs). The cross-tissue BMD pattern for lacZ MF is similar to that observed for DNA adducts, and is consistent with an oral route-of-exposure and differences in tissue-specific metabolism and proliferation. The lacZ MF BMDs were significantly correlated with the tissue-matched adduct BMDs, demonstrating a consistent adduct conversion rate across tissues. The BMD CIs, for both the Pig-a and the MN endpoints, overlapped for RETs and RBCs, suggesting comparable utility of both cell populations for protracted exposures. Examination of endpoint-specific response maxima illustrates the difficulty of comparing BMD values for a fixed benchmark response across endpoints. Overall, the BMD-approach permitted robust comparisons of responses across tissues/endpoints, which is valuable to our mechanistic understanding of how benzo[a]pyrene induces genetic damage.

Toxicological evaluation of Microbacterium foliorum SYG27B-MF.

Microbacterium foliorum is a naturally occurring bacteria in cruciferous vegetables and ripened cheese. The safety of M. foliorum SYG27B-MF has been assessed in both acute and subchronic studies and a battery of mutagenicity and clastogenicity tests. In a single dose acute study, the LD50 of M. foliorum SYG27B-MF was greater than 3 g/kg bw or 5.1 × 1016 colony forming unit (CFU)/kg bw, the highest dose tested. In a 90-day subchronic toxicity study in 80 Sprague-Dawley rats, no animals died and there were no treatment-related abnormalities at doses of 0, 500, 1000, or 2000 mg/kg bw. In a 90-day repeated toxicity test, the no-observed-adverse-effect level (NOAEL) M. foliorum SYG27B-MF was 2000 mg/kg/day or 3.4 × 1016 CFU/kg bw/day, the highest level tested. A mutagenicity study using reverse bacterial mutation tests and a genotoxicity study employing cultured hamster ovarian fibroblasts (CHO-K1) cell showed that M. foliorum SYG27B-MF was not mutagenic or clastogenic in the presence or absence metabolic activation. In an in vivo mouse micronucleus assay, M. foliorum SYG27B-MF did not induce did not induce micronuclei formation in the bone marrow cells of mice, indicating that it is non-clastogenic. The results from these studies support the safety of M. foliorum SYG27B-MF for use as a production organism for human food ingredients.

Toxicological evaluation of 3'-sialyllactose sodium salt.

The safety of 3'-sialyllactose (3'-SL) sodium salt was evaluated by testing for gene mutations, in vivo and in vitro clastogenic activity, and animal toxicity in beagle dogs and rats. The results of all mutagenicity and genotoxicity tests were negative, indicating that 3'-SL does not have any mutagenic or clastogenic potential. The mean lethal dose (LD50) of 3'-SL sodium salt was well above 20 g/kg body weight (bw) in rats. A dose escalation acute toxicity study in Beagle dogs also indicated no treatment-related abnormalities. Subsequent 28-day and 90-day toxicity studies in Sprague- Dawley (SD) rats involved dietary exposure to 500, 1,000, and 2000 mg/kg bw of 3'-SL sodium salt and a water (vehicle) control. There were no treatment-related abnormalities on clinical observations, body weight, food consumption, behavior, hematology, clinical chemistry, organ weights, relative organ weights, urinalysis parameters, or necropsy and histopathological findings. The No Observed Adverse Effect Level (NOAEL) of 3'-SL sodium salt was determined to be higher than 2000 mg/kg bw/day in an oral subchronic toxicity study in rats, indicating that the substance is an ordinary carbohydrate with the lowest toxicity rating. Results confirm that 3'-SL sodium salt has a toxicity profile similar to other non-digestible carbohydrates and naturally occurring human milk oligosaccharides (HMOs) and support its safety for human consumption in foods.

Cytotoxicity, mutagenicity, and antimutagenicity of the gentisic acid on HTC cells.

Gentisic acid (GA) exhibits antioxidant, anti-inflammatory, and antibiotic activities. This substance can be found in citrus fruits, grapes, olive oil, and peas. Considering that there are few studies in the literature on the toxicity of GA, the present work aimed to investigate its cytotoxic, mutagenic, and antimutagenic activities on HTC cells. GA was diluted in culture medium at the final concentration of 0.08, 0.16, 0.8, 1.6, and 8 µg/mL. The cytotoxicity was determined by the MTT assay and Trypan Blue exclusion method, with methyl methanesulfonate and doxorubicin as positive controls, respectively. The cytokinesis-block micronucleus assay determined the mutagenic/antimutagenic activity with benzo[a]pyrene as positive control. Negative control received culture medium only. GA (0.08-8 µg/mL) was not cytotoxic to HTC cells by the MTT assay nor the Trypan Blue exclusion method as no statistical difference was observed when compared to the control. Concentration of 0.08 and 0.8 µg/mL showed no mutagenic or clastogenic effects, as no significant micronuclei inductions were observed, different from 8 µg/mL, that was mutagenic. Furthermore, none of the concentrations presented an antiproliferative activity. The antimutagenic activity of GA (0.08 µg/mL) was observed at the simultaneous treatment, as it reduced the frequency of micronuclei by 76% (24 h) and 79% (48 h). Although pre- and post-treatments were not statistically different from the mutagen, they reduced the induced-damage by 11% and 21%, respectively. The present study indicated the absence of cytotoxicity and antiproliferative activities of GA, in addition to their antimutagenic/protective effects that may contribute to human health.

50% Ethanol extract of Orthosiphon stamineus modulates genotoxicity and clastogenicity induced by mitomycin C.

Herbal products contain a variety of compounds which may be useful in protecting against cellular damage caused by mutagens. Orthosiphon stamineus (O.s) also known as Cat whiskers. The herb has been shown anti-oxidative properties and can modulate key cellular proteins that have cytoprotective effect. The study aimed to evaluate the effects of different doses (250, 500 and 1000 mg kg-1) of 50% ethanol extract of O.s (Et. O.s) on micro-nucleated polychromatic erythrocytes (MNPCE), Polychromatic to normachromatic erythrocytes ratio (PCE/NCE), Mitotic index (MI), and Chromosomal aberration (CA) in Bab/c mice. Moreover, these parameters were used to evaluate the anti-genotoxic and clastogenic potencies of (Et. O.s) against mitomycin c (MMC) that interact with biological molecules and induce genotoxic and clastogenic disorders in non-tumor cells. MMC (4 mg kg-1) was injected intraperitoneally (i.p.) to the mice before and after treatment with three different doses of (Et. O.s). The results indicated that the extract at different doses did not show significant (p ≥ 0.05) differences in (MNPCE), (PCE/NCE) ratios, and (CA) values. The higher doses sowed high (MI) values compared with untreated control group. MMC showed significant increase (p ≤ 0.001) in (MNPCE), (CA) and reduce (PCE/NCE) and (MI) values compared with untreated control group. Treatment with (Et. O.s) at different doses before and after MMC injection showed to modulate MNPCE, PCE/NCE ratios, CA and MI values in mice bone marrow cells suggesting genoprotective potential of this plant extract.

New studies on the in vitro genotoxicity of sodium molybdate and their impact on the overall assessment of the genotoxicity of molybdenum substances.

The potential of molybdenum substances to cause genotoxic effects has been studied previously. However, a review of existing in vitro data, including an assessment of relevance and reliability, has shown that inconsistent results have been observed in the past. To resolve the inconsistencies, new studies were performed with the highly soluble sodium molybdate dihydrate according to OECD test guidelines. In a bacterial reverse mutation assay sodium molybdate dihydrate did not induce reverse mutations in five strains of Salmonella typhimurium. No mutagenic or clastogenic effect was observed at the tk locus of L5178Y mouse lymphoma cells. In a micronucleus test in cultured human peripheral blood lymphocytes no clastogenic or aneugenic effects were seen. These results can be read across to other inorganic molybdenum substances, that all release the molybdate ion [MoO4]2- under physiological conditions as the only toxicologically relevant species. In summary, a weight of evidence assessment of all available in vitro data shows no evidence of genotoxicity of molybdenum substances.

An oxovanadium(IV) complex protects murine bone marrow cells against cisplatin-induced myelotoxicity and DNA damage.

Cisplatin (CDDP) is one of the first-line anticancer drugs that has gained widespread use against various forms of human malignancies. But, the therapeutic outcome of CDDP therapy is limited due to its adverse effects including myelotoxicity and DNA damage which may lead to the subsequent risk of developing secondary cancer. Hence, in search of a suitable cytoprotectant, this study investigated the probable protective efficacy of an oxovanadium(IV) complex, namely oxovanadium(IV)-L-cysteine methyl ester complex (VC-IV) against CDDP-induced myelosuppression and genotoxic damage in the bone marrow cells of Swiss albino mice. CDDP was administered intraperitoneally (5 mg/kg b.w.) and VC-IV was administered orally (1 mg/kg b.w.) in concomitant and 7 d pretreatment schedule. Treatment with VC-IV in CDDP-treated mice significantly (p < 0.01) enhanced bone marrow cell proliferation and inhibited cell death in the bone marrow niche indicating improvement of CDDP-induced myelotoxicity. The organovanadium compound also significantly (p < 0.01) reduced the percentage of chromosomal aberrations, the frequency of micronuclei formation, and the extent of DNA damage. The observed chemoprotective effect of VC-IV was attributed to its anti-oxidant efficacy which significantly (p < 0.01) attenuated CDDP-induced generation of free radicals, and restored (p < 0.01) the levels of oxidized and reduced glutathione. Hence, VC-IV may serve as a promising candidate for future development to decrease the deleterious effects of CDDP in the bone marrow cells of cancer patients and associated secondary complications.

Review of genotoxicity and rat carcinogenicity investigations with astaxanthin.

Synthetic astaxanthin has been extensively tested for safety. Genotoxicity studies including Ames and in vitro Micronucleus Tests show absence of genotoxic potential. Although a long-term mouse study showed no carcinogenicity potential, the rat carcinogenicity study with dietary dosages of 0 (control), 0 (placebo beadlet), 40, 200 and 1000 mg astaxanthin/kg bw/day showed an increased incidence of benign, hepatocellular adenoma in females only, at 200 mg/kg bw/day and above. There was no clear evidence of toxicity during the in-life phase. Discoloration of feces was observed and a reduction in body weight gain in all groups receiving beadlets, probably reflecting a nutritional influence. Blood sampling confirmed systemic exposure and some minor clinical chemistry differences in females at 200 and 1000 mg/kg bw/day. There was no effect on adjusted liver weight. Histopathological examination showed hepatic changes indicative of slight hepatotoxicity and hepatocyte regeneration in females at 200 and 1000 mg/kg bw/day, in addition to the adenoma. Taking into account this pathological background in the female rat, and a wide variety of other supporting information, it is concluded that the hepatocellular adenoma in female rats was secondary to hepatotoxicity and regeneration, and is most probably a species-specific phenomenon of doubtful human relevance.

Evaluation of vinyl laurate in a battery of in vitro and in vivo tests for genotoxicity.

Vinyl laurate is a potential residual monomer in chewing gum base formulated with polyvinyl acetate vinyl laurate copolymer (PVAcVL). The genotoxic potential of vinyl laurate was examined in a battery of in vitro and in vivo genotoxicity tests. Vinyl laurate was not mutagenic in Ames tests. In addition, it was not mutagenic in the HPRT mutation assay in L5178Y cells. An in vitro mammalian chromosome aberration assay performed in CHO cells was equivocal. Vinyl laurate and/or its metabolites were not clastogenic in the mouse bone marrow micronucleus test. Kinetic data indicate that VL is metabolised to acetaldehyde and lauric acid. Both metabolites are well known and have been studied previously. Model calculations show, that any exposure to acetaldehyde from the consumption of PVAcVL containing chewing gum will remain far below levels of acetaldehyde exposure from food in which acetaldehyde occurs naturally. Direct exposure to VL will primarily be at the site of entry. The lack of toxicity in a 90-day repeated dose toxicity test, performed with VL doses up to approximately 3000 times higher than the maximal VL intake from the consumption of a typical piece of chewing gum, demonstrates a high safety margin.

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.

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.

Mutagenicity and safety evaluation of Ashwagandha (Withania somnifera) root aqueous extract in different models.

Withania somnifera (Ashwagandha) also called as Indian ginseng, a revered herb from Indian traditional system of medicine is a rejuvenator and tonic (Rasayana) used for its varied benefits. The roots of ashwagandha exhibit properties like anti-inflammatory, aphrodisiac, anthelmintic, astringent, diuretic, stimulant and thermogenic. However, data of ashwagandha on its mutagenic effects are lacking. In the present study, in-vitro genotoxicity tests were used to evaluate the mutagenic potential of Ashwagandha Root Extract (ARE). Concentrations of 0.156 to 5.00 mg/plate ARE were used for conducting Bacterial reverse mutation test (BRMT). For chromosome aberration (CA) test ARE was used in concentrations of 0.25 to 2.00 mg/ml, and for micronucleus (MN) tests ARE concentrations of 500/1000/2000 mg/kg were used. Acute oral toxicity was conducted in Wistar rats (n = 25) as per the OECD guideline (#423) with doses of 500/1000/2000 mg/kg body weight in male Swiss albino mice for morbidity and mortality for 3 days. The BRMT and CA tests were conducted with and without metabolic activation (S9). The study was approved by the institutional ethics committee (IEC) and institutional animal ethics committee (IAEC). ARE failed to show any mutagenic effects up to a dose of 5 mg/plate in BRMT. Also, ARE did not show any clastogenic activity in doses up to 2 mg/ml in CA test and in micronucleus test up to 2000 mg/kg body weight. These results were observed with and without metabolic activation (S9) under the stated experimental conditions. No mortality, morbidity, or any clinical signs were observed up to 3 days following ARE administration. Ashwagandha root extract failed to show any mortality in doses up to 2000 mg/kg oral dosage and did not show any mutagenic (genotoxic) effects in high concentrations.

Specific human CYP enzymes-dependent mutagenicity of tris(2-butoxyethyl) phosphate (an organophosphorus flame retardant) in human and hamster cell lines.

Tris(2-butoxyethyl) phosphate (TBOEP) is an organophosphorus flame retardant ubiquitously present in the environment and even the human body. TBOEP is toxic in multiple tissues, which forms dealkylated and hydroxylated metabolites under incubation with human hepatic microsomes; however, the impact of TBOEP metabolism on its toxicity, particularly mutagenicity (typically requiring metabolic activation), is left unidentified. In this study, the mutagenicity of TBOEP in human hepatoma cell lines (HepG2 and C3A) and the role of specific CYPs were studied. Through molecular docking, TBOEP bound to human CYP1A1, 1B1, 2B6 and 3A4 with energies and conformations favorable for catalyzing reactions, while the conformations of its binding with human CYP1A2 and 2E1 appeared unfavorable. In C3A cells (endogenous CYPs being substantial), TBOEP exposing for 72 h (2-cell cycle) at low micromolar levels induced micronucleus, which was abolished by 1-aminobenzotriazole (inhibitor of CYPs); in HepG2 cells (CYPs being insufficient) TBOEP did not induce micronucleus, whose effect was however potentiated by pretreating the cells with PCB126 (CYP1A1 inducer) or rifampicin (CYP3A4 inducer). TBOEP induced micronucleus in Chinese hamster V79-derived cell lines genetically engineered for stably expressing human CYP1A1 and 3A4, but not in cells expressing the other CYPs. In C3A cells, TBOEP selectively induced centromere protein B-free micronucleus (visualized by immunofluorescence) and PIG-A gene mutations, and elevated γ-H2AX rather than p-H3 (by Western blot) which indicated specific double-strand DNA breaks. Therefore, this study suggests that TBOEP may induce DNA/chromosome breaks and gene mutations in human cells, which requires metabolic activation by CYPs, primarily CYP1A1 and 3A4.

Transplacental clastogenic and epigenetic effects of gold nanoparticles in mice.

The broad application of nanotechnology in medicine, biology, and pharmacology is leading to a dramatic increase of the risk of direct contact of nanoproducts, among which gold nanoparticles (AuNP), with the human organism. The present study aimed at evaluating in vivo the genotoxicity of AuNPs with average size of 40 nm and 100 nm. A single intraperitoneal treatment of adult male and female Swiss mice (strain H) with AuNPs, at a dose of 3.3 mg/kg body weight, had no effect on the frequency of micronucleated polychromatic erythrocytes (MN PCEs) in bone marrow. Conversely, the transplacental treatment with AuNP-100 nm, but not with AuNP-40 nm, applied intraperitoneally at a dose of 3.3 mg/kg to pregnant mice on days 10, 12, 14, and 17 of gestation, resulted in a significant increase in the frequency of MN PCEs in both liver and peripheral blood of mouse fetuses. In parallel, the same treatment with AuNP-100 nm, but not with AuNP-40 nm, produced significant changes in microRNA expression. In particular, out of 1281 mouse microRNAs analyzed, 28 were dys-regulated more than two-fold and to a statistically significant extent in fetus lung, and 5 were up-regulated in fetal liver. Let-7a and miR-183 were significantly up-regulated in both organs. The data presented herein demonstrate for the first time the transplacental size-dependent clastogenic and epigenetic effects of AuNPs in mouse fetus, thus highlighting new aspects concerning the putative genotoxicity of AuNPs during a vulnerable period of life.