Cytoplasmic DNA: sources, sensing, and role in aging and disease.

Endogenous cytoplasmic DNA (cytoDNA) species are emerging as key mediators of inflammation in diverse physiological and pathological contexts. Although the role of endogenous cytoDNA in innate immune activation is well established, the cytoDNA species themselves are often poorly characterized and difficult to distinguish, and their mechanisms of formation, scope of function and contribution to disease are incompletely understood. Here, we summarize current knowledge in this rapidly progressing field with emphases on similarities and differences between distinct cytoDNAs, their underlying molecular mechanisms of formation and function, interactions between cytoDNA pathways, and therapeutic opportunities in the treatment of age-associated diseases.

ATR promotes clearance of damaged DNA and damaged cells by rupturing micronuclei.

The human ataxia telangiectasia mutated and Rad3-related (ATR) kinase functions in the nucleus to protect genomic integrity. Micronuclei (MN) arise from genomic and chromosomal instability and cause aneuploidy and chromothripsis, but how MN are removed is poorly understood. Here, we show that ATR is active in MN and promotes their rupture in S phase by phosphorylating Lamin A/C at Ser395, which primes Ser392 for CDK1 phosphorylation and destabilizes the MN envelope. In cells harboring MN, ATR or CDK1 inhibition reduces MN rupture. Consequently, ATR inhibitor (ATRi) diminishes activation of the cytoplasmic DNA sensor cGAS and compromises cGAS-dependent autophagosome accumulation in MN and clearance of micronuclear DNA. Furthermore, ATRi reduces cGAS-mediated senescence and killing of MN-bearing cancer cells by natural killer cells. Thus, in addition to the canonical ATR signaling pathway, an ATR-CDK1-Lamin A/C axis promotes MN rupture to clear damaged DNA and cells, protecting the genome in cell populations through unexpected cell-autonomous and cell-non-autonomous mechanisms.

Chromatin bridges: stochastic breakage or regulated resolution?

Genetic material is organized in the form of chromosomes, which need to be segregated accurately into two daughter cells in each cell cycle. However, chromosome fusion or the presence of unresolved interchromosomal linkages lead to the formation of chromatin bridges, which can induce DNA lesions and genome instability. Persistent chromatin bridges are trapped in the cleavage furrow and are broken at or after abscission, the final step of cytokinesis. In this review, we focus on recent progress in understanding the mechanism of bridge breakage and resolution. We discuss the molecular machinery and enzymes that have been implicated in the breakage and processing of bridge DNA. In addition, we outline both the immediate outcomes and genomic consequences induced by bridge breakage.

MITE infestation accommodated by genome editing in the germline genome of the ciliate Blepharisma.

During their development following sexual conjugation, ciliates excise numerous internal eliminated sequences (IESs) from a copy of the germline genome to produce the functional somatic genome. Most IESs are thought to have originated from transposons, but the presumed homology is often obscured by sequence decay. To obtain more representative perspectives on the nature of IESs and ciliate genome editing, we assembled 40,000 IESs of Blepharisma stoltei, a species belonging to a lineage (Heterotrichea) that diverged early from those of the intensively studied model ciliate species. About a quarter of IESs were short (<115 bp), largely nonrepetitive, and with a pronounced ~10 bp periodicity in length; the remainder were longer (up to 7 kbp) and nonperiodic and contained abundant interspersed repeats. Contrary to the expectation from current models, the assembled Blepharisma germline genome encodes few transposases. Instead, its most abundant repeat (8,000 copies) is a Miniature Inverted-repeat Transposable Element (MITE), apparently a deletion derivative of a germline-limited Pogo-family transposon. We hypothesize that MITEs are an important source of IESs whose proliferation is eventually self-limiting and that rather than defending the germline genomes against mobile elements, transposase domestication actually facilitates the accumulation of junk DNA.

A matter of wrapper: Defects in the nuclear envelope of lagging and bridging chromatin threatens genome integrity.

The nuclear envelope surrounds the eukaryotic genome and, through the nuclear pore complexes, regulates transport in and out of the nucleus. Correct nucleo-cytoplasm compartmentations are essential for nuclear functions such as DNA replication or repair. During metazoan mitosis, the nuclear envelope disintegrates to allow the segregation of the two copies of DNA between daughter cells. At the end of mitosis, it reforms on each group of chromosomes in the daughter cells. However, nuclear envelope reformation is delayed on lagging chromosomes and DNA bridges. Defects in the coordination between nuclear envelope reformation and chromosome segregation impair the nuclear functions. Mechanical stress to which micronuclei and DNA bridges are subjected to combined with their particular architecture and the altered nuclear functions result in DNA damage. While micronuclei and DNA bridges were considered for more than 100 years as mere indicators of chromosomal instability, rapid technological advances are helping to better understand the biological consequences of these aberrant nuclear morphologies. Recent studies provide interesting evidence that micronuclei and chromatin bridges act as a key platforms for a catastrophic mutational process observed in cancers called chromothripsis and a trigger for the innate immune response. Therefore, they could affect cellular functions by both genetic and non-genetic means.

Genome instability from nuclear catastrophe and DNA damage.

The nuclear envelope compartmentalizes the eukaryotic genome, provides mechanical resistance, and regulates access to the chromatin. However, recent studies have identified several conditions where the nuclear membrane ruptures during interphase, breaking down this compartmentalization leading to DNA damage, chromothripsis, and kataegis. This review discusses three major circumstances that promote nuclear membrane rupture, nuclear deformation, chromatin bridges, and micronucleation, and how each of these nuclear catastrophes results in DNA damage. In addition, we highlight recent studies that demonstrate a single chromosome missegregation can initiate a cascade of events that lead to accumulating damage and even multiple rounds of chromothripsis.

Chromoanagenesis, the mechanisms of a genomic chaos.

Designated under the name of chromoanagenesis, the phenomena of chromothripsis, chromanasynthesis and chromoplexy constitute new types of complex rearrangements, including many genomic alterations localized on a few chromosomal regions, and whose discovery over the last decade has changed our perception about the formation of chromosomal abnormalities and their etiology. Although exhibiting specific features, these new catastrophic mechanisms generally occur within a single cell cycle and their emergence is closely linked to genomic instability. Various non-exclusive exogenous or cellular mechanisms capable of generating chromoanagenesis have been evoked. However, recent experimental data shed light on 2 major processes, which following a defect in the mitotic segregation of chromosomes, can generate a cascade of cellular events leading to chromoanagenesis. These mechanisms are the formation of micronuclei integrating isolated chromosomal material, and the occurrence of chromatin bridges around chromosomal material resulting from telomeric fusions. In both cases, the cellular and molecular mechanisms of fragmentation, repair and transmission of damaged chromosomal material are consistent with the features of chromoanagenesis-related complex chromosomal rearrangements. In this review, we introduce each type of chromoanagenesis, and describe the experimental models that have allowed to validate the existence of chromoanagenesis events and to better understand their cellular mechanisms of formation and transmission, as well as their impact on the stability and the plasticity of the genome.

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.

An integrated in vitro carcinogenicity test that distinguishes between genotoxic carcinogens, non-genotoxic carcinogens, and non-carcinogens.

Chemical safety testing plays a crucial role in product and pharmacological development, as well as chemoprevention; however, in vitro genotoxicity safety tests do not always accurately predict the chemicals that will be in vivo carcinogens. If chemicals test positive in vitro for genotoxicity but negative in vivo, this can contribute to unnecessary testing in animals used to confirm erroneous in vitro positive results. Current in vitro tests typically evaluate only genotoxicity endpoints, which limits their potential to detect non-genotoxic carcinogens. The frequency of misleading in vitro positive results can be high, leading to a requirement for more informative in vitro tests. It is now recognized that multiple-endpoint genotoxicity testing may aid more accurate detection of carcinogens and non-carcinogens. The objective of this review was to evaluate the utility of our novel, multiple-endpoint in vitro test, which uses multiple cancer-relevant endpoints to predict carcinogenic potential. The tool assessed micronucleus frequency, p53 expression, p21 expression, mitochondrial respiration, cell cycle abnormalities and, uniquely, cell morphology changes in human lymphoblastoid cell lines, TK6 and MCL-5. The endpoints were used to observe cellular responses to 18 chemicals within the following categories: genotoxic carcinogens, non-genotoxic carcinogens, toxic non-carcinogens, and misleading in vitro positive and negative agents. The number of endpoints significantly altered for each chemical was considered, alongside the holistic Integrated Signature of Carcinogenicity score, derived from the sum of fold changes for all endpoints. Following the calculation of an overall score from these measures, carcinogens exhibited greater potency than non-carcinogens. Genotoxic carcinogens were generally more potent than non-genotoxic carcinogens. This novel approach therefore demonstrated potential for correctly predicting whether chemicals with unknown mechanism may be considered carcinogens. Overall, while further validation is recommended, the test demonstrates potential for the identification of carcinogenic compounds. Adoption of the approach could enable reduced animal use in carcinogenicity testing.

Low magnesium in conjunction with high homocysteine increases DNA damage in healthy middle aged Australians.

PURPOSE: Magnesium is one of the most common elements in the human body and plays an important role as a cofactor of enzymes required for DNA replication and repair and many other biochemical mechanisms including sensing and regulating one-carbon metabolism deficiencies. Low intake of magnesium can increase the risk of many diseases, in particular, chronic degenerative disorders. However, its role in prevention of DNA damage has not been studied fully in humans so far. Therefore, we tested the hypothesis that magnesium deficiency either on its own or in conjunction with high homocysteine (Hcy) induces DNA damage in vivo in humans. METHODS: The present study was carried out in 172 healthy middle aged subjects from South Australia. Blood levels of magnesium, Hcy, folate and vitamin B12 were measured. Cytokinesis-Block Micronucleus cytome assay was performed to measure three DNA damage biomarkers: micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) in peripheral blood lymphocytes. RESULTS: Data showed that magnesium and Hcy are significantly inversely correlated with each other (r = - 0.299, p < 0.0001). Furthermore, magnesium is positively correlated both with folate (p = 0.002) and vitamin B12 (p = 0.007). Magnesium is also significantly inversely correlated with MN (p < 0.0001) and NPB (p < 0.0001). Individuals with low magnesium and high Hcy exhibited significantly higher frequency of MN and NPBs compared to those with high magnesium and low Hcy (p < 0.0001). Furthermore, there was an interactive effect between these two factors as well in inducing MN (p = 0.01) and NPB (p = 0.048). CONCLUSIONS: The results obtained in the present study indicate for the first time that low in vivo levels of magnesium either on its own or in the presence of high Hcy increases DNA damage as evident by higher frequencies of MN and NPBs.

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.

A multi-biomarker micronucleus assay using imaging flow cytometry.

Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for causing genetic damage contributing to cancer risk in humans. Despite this, in vitro tests generate a high number of misleading positives, the consequences of which can lead to unnecessary animal testing and/or the abandonment of promising drug candidates. Understanding chemical Mode of Action (MoA) is vital to identifying the true genotoxic potential of substances and, therefore, the risk translation into the clinic. Here we demonstrate a simple, robust protocol for staining fixed, human-lymphoblast p53 proficient TK6 cells with antibodies against É£H2AX, p53 and pH3S28 along with DRAQ5™ DNA staining that enables analysis of un-lysed cells via microscopy approaches such as imaging flow cytometry. Here, we used the Cytek® Amnis® ImageStream®X Mk II which provides a high-throughput acquisition platform with the sensitivity of flow cytometry and spatial morphological information associated with microscopy. Using the ImageStream manufacturer's software (IDEAS® 6.2), a masking strategy was developed to automatically detect and quantify micronucleus events (MN) and characterise biomarker populations. The gating strategy developed enables the generation of a template capable of automatically batch processing data files quantifying cell-cycle, MN, É£H2AX, p53 and pH3 populations simultaneously. In this way, we demonstrate how a multiplex system enables DNA damage assessment alongside MN identification using un-lysed cells on the imaging flow cytometry platform. As a proof-of-concept, we use the tool chemicals carbendazim and methyl methanesulphonate (MMS) to demonstrate the assay's ability to correctly identify clastogenic or aneugenic MoAs using the biomarker profiles established.

Impact of indoor air pollution on DNA damage and chromosome stability: a systematic review.

Indoor air pollution is becoming a rising public health problem and is largely resulting from the burning of solid fuels and heating in households. Burning these fuels produces harmful compounds, such as particulate matter regarded as a major health risk, particularly affecting the onset and exacerbation of respiratory diseases. As exposure to polluted indoor air can cause DNA damage including DNA sd breaks as well as chromosomal damage, in this paper, we aim to provide an overview of the impact of indoor air pollution on DNA damage and genome stability by reviewing the scientific papers that have used the comet, micronucleus, and γ-H2AX assays. These methods are valuable tools in human biomonitoring and for studying the mechanisms of action of various pollutants, and are readily used for the assessment of primary DNA damage and genome instability induced by air pollutants by measuring different aspects of DNA and chromosomal damage. Based on our search, in selected studies (in vitro, animal models, and human biomonitoring), we found generally higher levels of DNA strand breaks and chromosomal damage due to indoor air pollutants compared to matched control or unexposed groups. In summary, our systematic review reveals the importance of the comet, micronucleus, and γ-H2AX assays as sensitive tools for the evaluation of DNA and genome damaging potential of different indoor air pollutants. Additionally, research in this particular direction is warranted since little is still known about the level of indoor air pollution in households or public buildings and its impact on genetic material. Future studies should focus on research investigating the possible impact of indoor air pollutants in complex mixtures on the genome and relate pollutants to possible health outcomes.

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.

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.

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.

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.

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.

Genotoxicity of bisphenol AF in rats: Detrimental to male reproductive system and probable stronger micronucleus induction potency than BPA.

Bisphenol AF (BPAF), as one of structural analogs of BPA, has been increasingly used in recent years. However, limited studies have suggested its adverse effects similar to or higher than BPA. In order to explore the general toxicity and genotoxicity of subacute exposure to BPAF, the novel 28-day multi-endpoint (Pig-a assay + micronucleus [MN] test + comet assay) genotoxicity evaluation platform was applied. Male rats were randomly distributed into seven main experimental groups and four satellite groups. The main experimental groups included BPAF-treated groups (0.5, 5, and 50 µg/kg·bw/d), BPA group (10 µg/kg·bw/d), two solvent control groups (PBS and 0.1% ethanol/99.9% oil), and one positive control group (N-ethyl-N-nitrosourea, 40 mg/kg bw). The satellite groups included BPAF high-dose recovery group (BPAF-HR), oil recovery group (oil-R), ENU recovery group (ENU-R), and PBS recovery group (PBS-R). All groups received the agents orally via gavage for 28 consecutive days, and satellite groups were given a recovery period of 35 days. Among all histopathologically examined organs, testis and epididymis damage was noticed, which was further manifested as blood-testis barrier (BTB) junction protein (Connexin 43 and Occludin) destruction. BPAF can induce micronucleus production and DNA damage, but the genotoxic injury can be repaired after the recovery period. The expression of DNA repair gene OGG1 was downregulated by BPAF. To summarize, under the design of this experiment, male reproductive toxicity of BPAF was noticed, which is similar to that of BPA, but its ability to induce micronucleus production may be stronger than that of BPA.