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Mutation Research. Genetic Toxicology... Apr 2024The risk of generating false positive in vivo comet assay results can be increased when procedural bias and/or technical variability is poorly controlled. This has been...
The risk of generating false positive in vivo comet assay results can be increased when procedural bias and/or technical variability is poorly controlled. This has been an ongoing concern since comet was first introduced into regulatory safety testing. But the proprietary nature of regulated studies and the 3Rs have limited the ability to conduct and publish the comparative in vivo studies necessary to determine the effect these factors can have on comet assay results when substances other than well characterized positive control compounds are evaluated in multiple tissues. That changed when Helix3 was asked to repeat for regulatory submission three independent in vivo comet studies with positive results generated by three other laboratories evaluating the effects of three different test substances on the liver, duodenum, and stomach. We repeated each study using the same test substance and experimental design as the original labs but with our standard quality control methods implemented to reduce procedural bias and variability. In every case, we generated negative results that regulatory authorities accepted over the initial positive results due to evidence of high technical variability and procedural bias in the original labs and studies. Meanwhile, the International Workshop on Genotoxicity (IWGT) compared >14 years of Helix3 comet historical control data (HCD) to HCD from 6 other experienced comet laboratories and concluded that our data exhibited the highest overall background % tail DNA levels with the lowest inter-study variability resulting in the highest quality HCD of all the labs evaluated. These case studies and the IWGT report suggest that our enhanced quality control methods and higher (>2 % mean of slide median tail DNA) background levels can effectively mitigate the nuisance factors that can generate false positive in vivo comet assay results. To facilitate a better understanding of the technical parameters that can significantly influence the comet results, we describe our enhanced procedures with justifications and examples.
Topics: Comet Assay; Reproducibility of Results; DNA Damage; Research Design; DNA
PubMed: 38575249
DOI: 10.1016/j.mrgentox.2024.503750 -
Mutation Research. Genetic Toxicology... Apr 2024'Modern' oral tobacco-free nicotine pouches (NPs) are a nicotine containing product similar in appearance and concept to Swedish snus. A three-step approach was taken to...
'Modern' oral tobacco-free nicotine pouches (NPs) are a nicotine containing product similar in appearance and concept to Swedish snus. A three-step approach was taken to analyse the biological effects of NPs and snus extracts in vitro. ToxTracker was used to screen for biomarkers for oxidative stress, cell stress, protein damage and DNA damage. Cytotoxicity, mutagenicity, and genotoxicity were assessed in the following respective assays: Neutral Red Uptake (NRU), Ames and Mouse Lymphoma Assay (MLA). Targeted analysis of phosphorylation signalling and inflammatory markers under non-toxic conditions was used to investigate any potential signalling pathways or inflammatory response. A reference snus (CRP1.1) and four NPs with various flavours and nicotine strengths were assessed. Test article extracts was generated by incubating one pouch in 20 mL of media (specific to each assay) with the inclusion of the pouch material. NP extracts did not induce any cytotoxicity or mutagenic response, genotoxic response was minimal and limited signalling or inflammatory markers were induced. In contrast, CRP1.1 induced a positive response in four toxicological endpoints in the absence of S9: Srxn1 (oxidative stress), Btg2 (cell stress), Ddit3 (protein damage) and Rtkn (DNA damage), and three endpoints in presence of S9: Srxn1, Ddit3 and Rtkn. CRP1.1 was genotoxic when assessed in MLA and activated signalling pathways involved in proliferation and cellular stress and specifically induced phosphorylation of c-JUN, CREB1, p53, p38 MAPK and to a lesser extent AKT1S1, GSK3α/β, ERK1/2 and RSK1 in a dose-dependent manner. CRP 1.1 extracts resulted in the release of several inflammatory mediators including cytokines IL-1α, IL5, IL6, IL8, IL-1RA, MIF and TNF-β, receptor IL-2RA, and growth factors FGF-basic, VEGF and M-CSF. In conclusion these assays contribute to the weight of evidence assessment of the potential comparative health risks of NPs and snus.
Topics: Mice; Animals; Nicotine; Tobacco, Smokeless; Mutagens; Oxidative Stress
PubMed: 38575247
DOI: 10.1016/j.mrgentox.2024.503738 -
Mutation Research 2024Environmental and occupational exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with adverse health effects in humans. Uncertainty exists regarding the...
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.
Topics: Benzo(a)pyrene; DNA Damage; Pilot Projects; Animals; Urothelium; Chromosomal Instability; Humans; Swine; Micronucleus Tests; Dose-Response Relationship, Drug; Chromosome Aberrations; Urinary Bladder Neoplasms; Time Factors; Comet Assay; Cell Line, Tumor; Urinary Bladder
PubMed: 38569440
DOI: 10.1016/j.mrfmmm.2024.111855 -
Molecules (Basel, Switzerland) Mar 2024Ipê is a plant of the Bignoniaceae family. Among the compounds extracted from this tree, lapachol is notable because its structural modification allows the production...
Ipê is a plant of the Bignoniaceae family. Among the compounds extracted from this tree, lapachol is notable because its structural modification allows the production of β-lapachone, which has anticancer properties. The objective of this work was to test this hypothesis at a cellular level in vitro and assess its potential safety for use. The following tests were performed: MTT cell viability assay, apoptotic index determination, comet assay, and micronucleus test. The results showed that β-lapachone had a high cytotoxic capacity for all cell lines tested: ACP02 (gastric adenocarcinoma cells), MCF7 (breast carcinoma cells), HCT116 (colon cancer cells) and HEPG2 (hepatocellular carcinoma cells). Regarding genotoxicity, the exposure of cells to sublethal doses of β-lapachone induced DNA damage (assessed by the comet assay) and nuclear abnormalities, such as nucleoplasmic bridges and nuclear buds (assessed by the micronucleus test). All tested cell lines responded similarly to β-lapachone, except for ACP02 cells, which were relatively resistant to the cytotoxic effects of the compound in the MTT test. Our results collectively indicate that although β-lapachone showed antiproliferative activity against cancer cell lines, it also caused harmful effects in these cells, suggesting that the use of β-lapachone in treating cancer should be carried out with caution.
Topics: Humans; Apoptosis; Naphthoquinones; Antineoplastic Agents; DNA Damage; Colonic Neoplasms
PubMed: 38543031
DOI: 10.3390/molecules29061395 -
Ecotoxicology and Environmental Safety Apr 2024A risk assessment on the aquatic toxicity of the plant biostimulant strigolactone mimic (2-(4-methyl-5-oxo-2,5-dihydro-furan-2-yloxy)-benzo[de]isoquinoline-1,3-dione...
A risk assessment on the aquatic toxicity of the plant biostimulant strigolactone mimic (2-(4-methyl-5-oxo-2,5-dihydro-furan-2-yloxy)-benzo[de]isoquinoline-1,3-dione (SL-6) was performed using a suite of standardised bioassays representing different trophic groups and acute and chronic endpoints. In freshwater, three trophic groups of algae, crustacea and fish were used. Whilst in seawater, algae (unicellular and macroalgae), Crustacea and Mollusca were employed. In addition, the genotoxicity of SL-6 was determined with the comet assessment performed on unicellular marine algae, oysters, and fish embryos. This was the first time ecotoxicity tests have been performed on SL-6. In freshwater, the lowest LOEC was measured in the unicellular algae at 0.31 mg/L SL-6. Although, similar LOEC values were found for embryo malformations and impacts on hatching rate in zebrafish (LOEC 0.31-0.33 mg/L). Consistent malformations of pericardial and yolk sac oedemas were identified in the zebrafish embryos at 0.31 mg/L. In marine species, the lowest LOEC was found for both Tisbe battagliai mortality and microalgae growth at an SL-6 concentration of 1.0 mg/L. Significant genotoxicity was observed above control levels at 0.0031 mg/L SL-6 in the unicellular algae and 0.001 mg/L SL-6 in the oyster and zebrafish larvae. When applying the simple risk assessment, based on the lowest NOECs and appropriate assessment factors, the calculated predicted no effect concentration (PNEC), for the ecotoxicity and the genotoxicity tests were 1.0 µg/L and 0.01 µg/L respectively.
Topics: Animals; Zebrafish; Larva; Crustacea; Mutagenicity Tests; Water Pollutants, Chemical; Heterocyclic Compounds, 3-Ring; Lactones
PubMed: 38537480
DOI: 10.1016/j.ecoenv.2024.116244 -
Journal of Xenobiotics Dec 2023Triclosan and Triclocarban, preservatives widely used in cosmetics and other consumer products, underwent evaluation using a battery of new-approach methodologies in...
Triclosan and Triclocarban, preservatives widely used in cosmetics and other consumer products, underwent evaluation using a battery of new-approach methodologies in vitro (NAMs). Specifically, the Microplate Ames Test (MPF™ Test, Xenometrix, Allschwil, Switzerland) was employed to assess mutagenicity, the Comet assay in vitro on the HaCat cell line and the Mammalian Chromosome Aberration Test were utilized to evaluate genotoxicity, and the XenoScreen YES/YAS assay was applied to investigate endocrine disruption. The chemicals did not exhibit any positive responses for mutagenicity. However, the mammalian chromosome aberration test identified both chemicals as being positive for genotoxicity at 10 µg/mL. In the Comet assay, the percentage of DNA in the tail significantly increased in a concentration-dependent manner (at 5 and 10 µg/mL for Triclosan, at 2.5, 5, and 10 µg/mL for Triclocarban). The positive response depended on the increasing concentration and the duration of exposure. Triclosan, but not Triclocarban in any of the endocrine assays performed, indicated a potential for endocrine activity in the anti-estrogenic and anti-androgenic assays. The positive in vitro results detected were obtained for concentrations relevant to final products. The alarming findings obtained with the use of new-approach methodologies (NAMs) justify the current precautionary regulatory approach, limiting the use of these preservatives.
PubMed: 38535491
DOI: 10.3390/jox14010002 -
BMC Oral Health Mar 2024To analyze anti-MMP mode of action of Quaternary Ammonium Silane (QAS, codenamed as k21) by binding onto specific MMP site using computational molecular simulation and...
AIMS AND OBJECTIVES
To analyze anti-MMP mode of action of Quaternary Ammonium Silane (QAS, codenamed as k21) by binding onto specific MMP site using computational molecular simulation and Anti-Sortase A (SrtA) mode of action by binding onto specific site using computational molecular simulation.
MATERIALS AND METHODS
In silico Molecular Dynamics (MD) was used to determine the interactions of K21 inside the pocket of the targeted protein (crystal structure of fibroblast collagenase-1 complexed to a diphenyl-ether sulphone based hydroxamic acid; PDB ID: 966C; Crystal structure of MMP-2 active site mutant in complex with APP-derived decapeptide inhibitor. MD simulations were accomplished with the Desmond package in Schrödinger Drug Discovery Suite. Blood samples (~ 0.5 mL) collected into KEDTA were immediately transferred for further processing using the Litron MicroFlow® PLUS micronucleus analysis kit for mouse blood according to the manufacturer's instructions. Bacterial Reverse Mutation Test of K21 Molecule was performed to evaluate K21 and any possible metabolites for their potential to induce point mutations in amino acid-requiring strains of Escherichia coli (E. coli) (WP2 uvrA (tryptophan-deficient)).
RESULTS
Molecular Simulation depicted that K21 has a specific pocket binding on various MMPs and SrtA surfaces producing a classical clouting effect. K21 did not induce micronuclei, which are the result of chromosomal damage or damage to the mitotic apparatus, in the peripheral blood reticulocytes of male and female CD-1 mice when administered by oral gavage up to the maximum recommended dose of 2000 mg/kg. The test item, K21, was not mutagenic to Salmonella typhimurium (S. typhimurium) strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2 uvrA in the absence and presence of metabolic activation when tested up to the limit of cytotoxicity or solubility under the conditions of the test.
CONCLUSION
K21 could serve as a potent protease inhibitor maintaining the physical and biochemical properties of dental structures.
Topics: Mice; Male; Female; Animals; Mutagenicity Tests; Ammonium Compounds; Escherichia coli; Mutagens; Matrix Metalloproteinases
PubMed: 38528501
DOI: 10.1186/s12903-024-04069-0 -
Mutation Research. Reviews in Mutation... 2024Humans ingest particles and fibers on daily basis. Non-digestible carbohydrates are beneficial to health and food additives are considered safe. However, titanium... (Review)
Review
Humans ingest particles and fibers on daily basis. Non-digestible carbohydrates are beneficial to health and food additives are considered safe. However, titanium dioxide (E171) has been banned in the European Union because the European Food Safety Authority no longer considers it non-genotoxic. Ingestion of microplastics and nanoplastics are novel exposures; their potential hazardous effects to humans have been under the radar for many years. In this review, we have assessed the association between oral exposure to man-made particles/fibers and genotoxicity in gastrointestinal tract cells and secondary tissues. We identified a total of 137 studies on oral exposure to particles and fibers. This was reduced to 49 papers with sufficient quality and relevance, including exposures to asbestos, diesel exhaust particles, titanium dioxide, silver nanoparticles, zinc oxide, synthetic amorphous silica and certain other nanomaterials. Nineteen studies show positive results, 25 studies show null results, and 5 papers show equivocal results on genotoxicity. Recent studies seem to show null effects, whereas there is a higher proportion of positive genotoxicity results in early studies. Genotoxic effects seem to cluster in studies on diesel exhaust particles and titanium dioxide, whereas studies on silver nanoparticles, zinc oxide and synthetic amorphous silica seem to show mainly null effects. The most widely used genotoxic tests are the alkaline comet assay and micronucleus assay. There are relatively few results on genotoxicity using reliable measurements of oxidatively damaged DNA, DNA double strand breaks (γH2AX assay) and mutations. In general, evidence suggest that oral exposure to particles and fibers is associated with genotoxicity in animals.
Topics: DNA Damage; Animals; Gastrointestinal Tract; Humans; Titanium; Mutagenicity Tests
PubMed: 38522822
DOI: 10.1016/j.mrrev.2024.108491 -
Mutagenesis Apr 2024The 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...
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.
Topics: Micronucleus Tests; Humans; Animals; Nanostructures; Cricetinae; Cricetulus; Cell Line; Organisation for Economic Co-Operation and Development; Hep G2 Cells
PubMed: 38502821
DOI: 10.1093/mutage/geae010 -
Indian Journal of Pharmacology Jan 2024Cosmeceuticals are topically applied cosmetic products containing a biologically active ingredient with a pharmaceutical effect that improves, nourishes, and treats the... (Review)
Review
Cosmeceuticals are topically applied cosmetic products containing a biologically active ingredient with a pharmaceutical effect that improves, nourishes, and treats the skin appearance. The trend of cosmeceuticals began during the mid-20th century due to its potent ingredients with therapeutic effects for various skin ailments. Even though there is a great advancement in cosmetics, which shows the risk of cosmetic linked melanoma, endocrine disorders, and birth defects which was one in 1500 people during 1935 have increased to one in 75 people in 2000. Hence, as a part of reducing the harmful effect, natural ingredients were added to the formulation to give the pharmaceutical effect. Thus, natural/herbal cosmeceuticals were introduced. Due to the awareness of the side effects such as photo-toxicity, mutagenicity, irritation by these synthetic products, people started preferring herbal/natural cosmetic products. Moreover, natural cosmeceuticals were proven to be effective against various dermatological conditions as well as have fewer side effects marked the natural/herbal cosmeceuticals in the market. Unlike a drug, cosmeceutical products undergo safety, toxicity, and efficacy tests, but these are not classified under Food and Drug Administration. This review will give an insight into different natural ingredients used in natural/herbal cosmeceutical formulation and their function challenges faced during formulation, advantages of natural cosmeceuticals over regular cosmeceuticals, and regulatory aspects in India.
Topics: Humans; Cosmeceuticals; Cosmetics; Skin; Pharmaceutical Preparations; Pharmaceutical Vehicles; Biological Products
PubMed: 38454588
DOI: 10.4103/ijp.ijp_244_21