L5178Y Mouse Lymphoma Research Articles

Evaluation of the mutagenic effects of Molnupiravir and N4-hydroxycytidine in bacterial and mammalian cells by HiFi sequencing.

Molnupiravir (MOV) is used to treat COVID-19. In cells, MOV is converted to the ribonucleoside analog N4-hydroxycytidine (NHC) and incorporated into the SARS-CoV-2 RNA genome during its replication, resulting in RNA mutations. The widespread accumulation of such mutations inhibits SARS-CoV-2 propagation. Although safety assessments by many regulatory agencies across the world have concluded that the genotoxic risks associated with the clinical use of MOV are low, concerns remain that it could induce DNA mutations in patients, particularly because numerous in vitro studies have shown that NHC is a DNA mutagen. In this study, we used HiFi sequencing, a technique that can detect ultralow-frequency substitution mutations in whole genomes, to evaluate the mutagenic effects of MOV in E.coli and of MOV and NHC in mouse lymphoma L5178Y cells and human lymphoblastoid TK6 cells. In all models, exposure to these compounds increased genome-wide mutation frequencies in a dose-dependent manner, and these increases were mainly composed of A:T → G:C transitions. The NHC exposure concentrations used for mammalian cells were comparable to those observed in the plasma of humans who received clinical doses of MOV.

Thymidine Kinase+/- Mammalian Cell Mutagenicity Assays for Assessment of Nanomaterials.

The methods outlined here are part of a series of papers designed specifically for genotoxicity assessment of nanomaterials (NM). Common Considerations such as NM characterization, sample preparation and dose selection, relevant to all genotoxicity assays, are found in an accompanying paper. The present paper describes methods for evaluation of mutagenicity in the mammalian (mouse) thymidine kinase (Tk) gene occurring in L5178Y mouse lymphoma (ML) cells and in the designated TK gene in human lymphoblastoid TK6 cells. Mutations change the functional genotype from TK+/- to TK-/-, detectable as cells surviving on media selective for the lack of thymidine kinase (TK) function. Unlike cells with TK enzyme function, the TK-/- cells are unable to integrate the toxic selection agent, allowing these cells to survive as rare mutant colonies. The ML assay has been shown to detect a broad spectrum of genetic damage, including both small scale (point) mutations and chromosomal alterations. This assay is a widely used mammalian cell gene mutation assay for regulatory purposes and is included in the core battery of genotoxicity tests for regulatory decision-making. The TK6 assay is an assay using a human cell line derived similarly via mutagenic manipulations and optimal selection. Details are provided on the materials required, cell culture methods, selection of test chemical concentrations, cytotoxicity, treatment time, mutation expression, cloning, and data calculation and interpretation. The methods describe the microwell plate version of the assays without metabolic activation.

Genome-wide detection of ultralow-frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms by PacBio sequencing.

Many conventional genetic toxicology assays require specialized cell cultures or animals and can only detect mutations that inactivate the function of a reporter gene. These limitations make such assays incompatible with many toxicological models but could be overcome by the development of techniques capable of directly detecting genome-wide somatic mutations through DNA sequencing. PacBio sequencing can generate almost error-free consensus reads by repeatedly inspecting both DNA strands from circularized molecules (a method known as PacBio HiFi). In this study, we show that PacBio HiFi can detect genome-wide ultralow-frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms. The mutation frequencies (MFs) of unexposed samples in both models were ~1 × 10-7 mutations per base pair. Compared to these controls, PacBio HiFi detected MF increases of 23-fold in cultures of L5178Y cells exposed to 5 mM ethyl methanosulfonate (EMS) for 4h, and 5-, 12-, and 29-fold in cultures of C. elegans worms exposed to 12.5, 25, and 50 mM EMS for 4h, respectively. In both models, the mutation spectra of controls were diverse, while those derived from EMS-exposed samples were dominated by C:G → T:A transitions. To validate these results, clone sequencing analyses were performed on the same cultures of L5178Y cells. The results obtained by clone sequencing and PacBio HiFi were almost identical. Our results suggest that PacBio sequencing could be used for the detection, quantitation, and characterization of mutations in any DNA-containing sample, including those that are not compatible with conventional mutation detection approaches.

Mutagenicity test of Streptomyces Species culture filtrate using mouse lymphoma cells

Introduction: Streptomyces species culture was isolated and identified. Its filtrate was found to be able to damage Odontoglossum ringspot virus and Cymbidium mosaic virus coat protein in our previous study. It is absolutely necessary to establish its biohazard level and toxicological profile before this filtrate or its active compound can be used for immediate disinfection of the equipment and bench surface, or even technician’s skin. Aim: In this study we intend to test the mutagenicity of this culture filtrate using the mouse lymphoma assay (MLA). Material and methods: Streptomyces species was cultured for 14 days, and the medium was collected for XTT assay to determine the highest concentration of the culture filtrate that do not cause reduction in cell number. In vitro mammalian cell gene mutation test using Mouse lymphoma L5178Y TK+/- was used to evaluate the mutagenic potential of the culture filtrate in accordance with OECD (1997), test No. 476: in vitro Mammalian Cell Gene Mutation Test. Result: Our cytotoxicity assay revealed that 1.33% of the culture filtrate was the maximal concentration that would not inflict reduction in cell number. The result of mutation test showed that with 3-hour treatment and S9 metabolic activation, the culture filtrate did not show significant mutagenicity when compared to the negative controls (ANOVA and Student t-test, p<0.05). Summary: This data suggested that the culture filtrate is not mutagenic.

Structure-activity relationship study: Mechanism of cyto-genotoxicity of Nitropyrazole-derived high energy density materials family

Stringent toxicological tests have to be performed prior to the industrial development of alternative chemicals particularly high energy dense materials (HEDMs) such as explosives. The properties (e.g., power, stability) of these compounds are constantly being improved, the current axis of research being the nitration of nitrogen heterocycles leading to HEDMs such as nitropyrazole-derived molecules. However, except for 3,4,5-trinitropyrazole (3,4,5-TNP), which was shown to be highly toxic in mice, the toxicological impact of these HEDMs has so far not been investigated. Furthermore, as industrials are strongly advised to develop alternative safety testing assays to in vivo experiments, we herein focused on determining the cytotoxic and genotoxic effects of seven Nitropyrazole-derived HEDMs on three rodent cell lines (mouse embryonic BALB/3T3 clone A31 cells, Chinese hamster ovary cells CHO-K1 and mouse lymphoma L5178Y TK +/− clone (3.7.2C) cells), two human fibroblast lines (CRC05, PFS04062) and on the human hepatic HepaRG model (both in proliferative and differentiated cells). A stronger cytotoxic effect was observed for 1,3-dinitropyrazole (1, 3-DNP) and 3,4,5-TNP in all cell lines, though differentiated HepaRG cells clearly displayed fewer likely due to the metabolism and elimination of these molecules by their functional biotransformation pathways. At the mechanistic level, the sub-chronic cytotoxic and genotoxic effects were linked to ROS/RNS production (experimental assays), HA2.X and to transcriptomic data highlighting the increase in DNA repair mechanisms.

Genotoxicity evaluation of titanium dioxide nanoparticles using the mouse lymphoma assay and the Ames test

Titanium dioxide nanoparticles (TiO2-NPs) are widely used in the cosmetics, health, and food industries, but their safety and genotoxicity remain a matter of debate. We investigated whether TiO2-NPs could induce gene mutations in mouse lymphoma L5178Y cells and Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA1535. Following preliminary tests, 2 mg/mL for the mouse lymphoma gene mutation assay and 1.25 mg/plate for the in vitro bacterial reverse mutation assay (Ames test) were selected as the highest concentrations. Exposure to TiO2-NPs for 4 or 24 h with or without S9 metabolic activation did not increase mutation frequency for any of the concentrations tested in L5178Y cells. In the Ames test, TiO2-NPs did not induce reverse mutation in the bacterial strains. No positive mutagenic responses were observed in either test system, and therefore we cannot classify TiO2-NPs as mutagenic; further testing will be required to determine conclusively whether TiO2-NPs are genotoxic.

Toxicity and Metal Corrosion of Glutaraldehyde-Didecyldimethylammonium Bromide as a Disinfectant Agent.

The wide use of disinfectants has prompted resistance from the microbiome which will in turn reduce the bactericidal effect of disinfectants. Hence, glutaraldehyde (GA) and didecyldimethylammonium bromide (DDAB) were used to develop a combination disinfectant with high stability and antimicrobial effects, which was named GA-DDAB combination disinfectant (GD). The bactericidal mechanism against Escherichia coli was studied in our earlier work. In this study, we focused on GD's bactericidal efficacy in both the laboratory and environment, the genetic toxicity to mouse lymphoma L5178Y TK+/− cells, acute peroral toxicity in mice, and its metal corrosion properties with a view to providing theoretical support for developing a high-efficiency, low toxicity, and weakly corrosive disinfectant for general use.

Cytotoxic secondary metabolites from the endophytic fungus Aspergillus versicolor KU258497

Two new isocoumarin dimers (1 and 2) and one new dihydroquinolone derivative (3) were isolated from Aspergillus versicolor, an endophyte derived from leaves of the Egyptian water hyacinth Eichhornia crassipes (Pontederiaceae), together with ten other known metabolites. Chemical structures of the isolated metabolites were determined based on HRESIMS, extensive 1D and 2D NMR spectroscopy. The relative and absolute configurations of the new natural products were established by ROESY and electronic circular dichroism (ECD) spectroscopy, respectively. The axial chirality of the isocoumarin 7,7′-homodimers (1 and 2) was deduced by TDDFT-ECD calculations. All isolated compounds were assessed for their antimicrobial, antitubercular and cytotoxic activities. Several tested compounds revealed significant cytotoxic activity against mouse lymphoma L5178Y cell line with IC50 values ranging from <0.36 to 16.3 μM.

Chemical constituents from leaves and root bark of Trichilia monadelpha (Meliaceae)

Two new limonoid derivatives designated, monadelphin A (1) and monadelphin B (2) and two new sesquiterpene derivatives named trichins A (3) and B (4) were isolated together with six known compounds (5–10) from the mixture of methylene chloride/methanol (1:1) extract of leaves and root bark of Trichilia monadelpha (Meliaceae) collected in Cameroon. The structures of the new compounds were unambiguously established by detailed spectroscopic analysis including 1D and 2D NMR data in conjunction with high resolution mass spectrometry data and by comparison of these data with those of related compounds described in the literature. Compounds 1–4 were screened for their cytotoxic potential. Compound 1 showed strong cytotoxicity against the mouse lymphoma L5178Y cell line with an IC50 value of 0.62μg/mL. The biogenetic origin of trichin B (4) from trichin A (3) was also postulated.

Predicting in vitro genotoxicity by mouse lymphoma L5178Y thymidine kinase mutation assay (MLA): Accounting for simulated metabolic activation of chemicals

The mouse lymphoma L5178Y thymidine kinase locus gene mutation assay (MLA) is typically part of regulatory batteries of methods used for in vitro evaluation of substances eliciting small gene mutations, and gross structural alterations at the chromosomal level. In an effort to make the MLA endpoint amenable to category formation to support assessments of mutagenesis, a model with 52 DNA and 32 protein binding alerts particular to the MLA endpoint has been developed. Each alert is supported by a mechanistic justification, an alert-specific training set and an alert performance evaluation. Subsequently, these alerts have been used in combination with an in vitro rat liver S9 fraction metabolic simulator (the TIssue MEtabolism Simulator (TIMES) platform) to evaluate MLA mutagenicity of likely metabolites of MLA negative parent compounds. The resulting system provides for transparent in silico identification of structural and general parametric requirements, as well as, binding mechanisms in parent chemicals and their simulated metabolites. When applied to the training sets, performance of the TIMES_MLA models, both with and without rat liver S9 activation show high (≥80%) sensitivity, specificity and concordance, which is consistent with the repeatability and reproducibility of experimental MLA results.

Cytotoxic drimane meroterpenoids from the Indonesian marine sponge Dactylospongia elegans

Chemical investigation of the methanol extract of an Indonesian marine sponge Dactylospongia elegans (family Thorectidae) afforded two undescribed drimane meroterpenoidal metabolites, dactylospongenones G and H (1 and 2) together with thirteen known compounds (3–15) including pelorol, 5-epi-ilimaquinone, 5-epi-smenospongine, 5-epi-smenospongidine, nakijiquinone D, smenospongine C, isospongiaquinone, isosmenospongine, nakijiquinones A-C, G and 5-epi-nakijiquinone Q. The structures of the new metabolites were unambiguously determined by 1D and 2D NMR spectroscopy and by HRESIMS. All isolated compounds were assessed for their antimicrobial, antitubercular and cytotoxic activities. Several tested compounds revealed significant cytotoxic activity against the mouse lymphoma L5178Y cell line with IC50 values ranging from 1.3 to 6.5μM. In antimicrobial and antitubercular activity assays some of the tested compounds exhibited potent activity with MIC values between 3.0 and 25.0μM.

Colletotrin: a sesquiterpene lactone from the endophytic fungus Colletotrichum gloeosporioides associated with Trichilia monadelpha

Abstract A new sesquiterpene lactone, namely colletotrin (1), together with two known fungal metabolites (2, 3), was obtained from a rice culture of Colletotrichum gloeosporioides, an endophytic fungus isolated from the stem bark of Cameroonian medicinal plant Trichilia monadelpha (Meliaceae). The structure of the new compound was established on the basis of extensive NMR analysis (1H, 13C, heteronuclear single-quantum coherence and heteronuclear multiple-bond correlation) completed by high-resolution electrospray ionization mass spectroscopy results and by comparison of these data with those of related compounds described in the literature. Their cytotoxic and antibacterial activities against the mouse lymphoma L5178Y cell line and Mycobacterium tuberculosis, respectively, were also evaluated. This study is the first report on the isolation of fungal metabolites from endophytic fungi associated with T. monadelpha (Meliaceae).

Development of a quantitative cytotoxicity assay using mouse lymphoma TK cells

We describe a new, quantitative, cytotoxicity method using mouse lymphoma L5178Y thymidine kinase+/− (TK) suspension cells for medical device safety assessment that has equivalent sensitivity, and greater efficiency compared to the colony formation assay (CFA) using adherent V79-4 cells. Assays were carried out in 24-well plates. TK cells were seeded on concave side of six types of contact lenses and cell growth rate after 72h determined using the Vi-Cell XR. Five of six contact lenses studied demonstrated the same results between the TK and the CFA methods. One lens showed a difference between the CFA and TK suspension cell methods (79% colony formation rate vs. 17% cell growth rate). Although colony size is not a CFA test criterion, colonies were smaller than control indicating a cytotoxic effect that is reflected in the TK suspension cell assay which measures cell number. The mouse lymphoma TK suspension cell assay performs as well as the CFA direct contact method with distinct advantages. This quantitative, automated method is not susceptible to mechanical damage from test materials, does not depend on cell adherence to test materials and represents a valuable tool to evaluate medical device cytotoxicity.

Comparative toxicity related to metabolisms of benzophenone-type UV filters, potentially harmful to the environment and humans

Recently, sunscreens containing ultraviolet (UV)-blocking substance actively encourage to reduce damage caused by solar UV radiation. Benzophenone (BP)-type UV filters have become widely used as UV stabilizers in skin-moisturizing products and sunscreen lotions of human skin and hair. However, very little information is available regarding the potential harmful effects of long term exposure to these substances. Therefore, we investigated the possibility of genotoxic effects related to metabolism of BP-type UV filters in mouse lymphoma L5178Y cells. In metabolism process of BP-type UV filters, BP was mainly converted to benzhydrol (BH) and 4-hydroxybenzophenone (HBP) (i.e., type A UV filters). In contrast, HMB was converted into at least three intermediates, including 2,4-dihydroxybenzophenone (DHB), which was formed via o-demethylation and subsequently converted into 2,3,4-trihydroxybenzophenone (THB), and 2,2′-dihydroxy-4-methoxybenzophenone (DHMB), which formed via the aromatic hydroxylation of HMB (i.e., type B UV filters). The comet assay was used to assess DNA damage in type A and B UV filters-treated cells. We found that cells exposed to type A or B UV filters showed significant DNA damage compared to untreated cells. Exposure to HBP, the hydroxylated metabolite of BP, and DHB and THB, the di- and tri-hydroxylated metabolites of HMB, resulted in particularly severe DNA damage (P<0.05). Therefore, we suggest that BP-type UV filters with a hydroxyl group at the para-position (e.g., HBP, DHB, and THB) may induce single-stranded DNA breakage in L5178Y cells.

Cytotoxic labdane diterpenes and bisflavonoid atropisomers from leaves of Araucaria bidwillii

Chemical investigation of a methanolic extract of leaves from Araucaria bidwillii (Araucariaceae) from Egypt afforded four new labdane diterpenoidal metabolites (1–4) together with one known diterpene, 7-oxocallitrisic acid (5), two triterpenoidal metabolites, 2-O-acetyl-11-keto-boswellic acid (6) and β-sitosterol-3-O-glucopyranoside (7), phloretic acid (8), and two methylated bisflavonoids, agathisflavone-4′,7,7″-trimethyl ether (9) and cupressuflavone-4′,7,7″-trimethyl ether (10). The new metabolites 1–4 were unambiguously identified by applying extensive 1D and 2D NMR spectroscopic studies as well as HRESIMS. The relative and absolute configurations of 1–4 were determined using ROESY and the modified Mosher's method, respectively. All isolated compounds were assessed for their antimicrobial, antitubercular and cytotoxic activities. Among the tested compounds, the new labdane diterpenes 1–4 revealed significant cytotoxic activity against mouse lymphoma L5178Y cell line with IC50 values ranging from 1.4 to 12.9 μM, respectively.

Structural chromosome aberrations cause swelling of the nucleus.

BackgroundCarcinogens are known to cause swelling of the mammalian cell nucleus. However, the mechanism of the swelling and its toxicological significance have not been fully elucidated. Since nuclear swelling (NS hereafter) has been frequently observed in chromosomal aberration (CA hereafter) tests (in vitro), the relationship between NS and CAs was investigated in this study.ResultsIn a short-term CA test using the fibroblast CHL cell line, the appearance of NS increased in a dose-dependent manner after exposure to six types of clastogens (mitomycin C, methyl methane sulfonate, 1-methyl-3-nitro-1-nitrosoguanidine, benzo[a]pyrene, cyclophosphamide monohydrate, and 9,10-dimethyl-2-benzanthracene), and a strong correlation was found between NS (%) and CAs (%) at each dosage. Therefore, we hypothesized that clastogens cause NS in cultured mammalian cells, since the mouse lymphoma L5178Y cell line is known to have a similar sensitivity to clastogens. Thus, we measured NS for 14 compounds (clastogens) that are known to induce structural CAs, 4 aneugens, and 12 non-mutagenes. Almost all clastogens caused NS of more than 5 %, which increased in a dose-dependent manner. Among the aneugens, colchicine, and diethylstilbestrol caused the same level of NS % as the clastogens, while carbendazim and trichlorfon caused a similar level of NS % as the clastogens only at higher levels of cytotoxicity. Almost all the non-mutagens caused less than 5 % NS.ConclusionsThese results strongly suggest that NS is mainly caused by structural aberrations in the nucleus during interphase of the cell cycle.

Pestalotiopamide E and pestalotiopin B from an endophytic fungus Aureobasidium pullulans isolated from Aloe vera leaves

A detailed chemical investigation of the mycelial extract of the endophytic fungus Aureobasidium pullulans isolated from leaves of the Moroccan Aloe vera yielded one new amide pestalotiopamide E (1) and its corresponding new acid pestalotiopin B (2) together with two indole metabolites (3 and 4), isoochracinic acid (5) and two hydronaphthalene derivatives (6 and 7). All isolated compounds were tested for their antiproliferative activity against mouse lymphoma L5178Y cell line.

Inducing secondary metabolite production by the endophytic fungus Chaetomium sp. through fungal–bacterial co-culture and epigenetic modification

Co-culturing the fungal endophyte Chaetomium sp. with the bacterium Bacillus subtilis on solid rice medium resulted in an up to 8.3-fold increase in the accumulation of constitutively present metabolites that included a 1:1 mixture of 3- and 4-hydroxybenzoic acid methyl esters (1 and 2, respectively), and the polyketides acremonisol A (3), SB236050 (4), and SB238569 (5). In addition, seven compounds including isosulochrin (6), protocatechuic acid methyl ester (7), as well as five new natural products (8–12) were detected in the co-cultures, but not in axenic fungal cultures. Treatment of Chaetomium sp. with the epigenetic modifier suberoylanilide hydroxamic acid or 5-azacytidine resulted in an enhanced accumulation of 6, which was likewise detected during co-culture. Compound 5 showed strong cytotoxicity against the mouse lymphoma L5178Y cell line with an IC50 value of 1 μM, as well as weak antibacterial activity against B. subtilis with an MIC value of 53 μM.

The clastogenicity of 4NQO is cell-type dependent and linked to cytotoxicity, length of exposure and p53 proficiency.

4-Nitroquinoline 1-oxide (4NQO) is used as a positive control in various genotoxicity assays because of its known mutagenic and carcinogenic properties. The chemical is converted into 4-hydroxyaminoquinoline 1-oxide and gives rise to three main DNA adducts, N-(deoxyguanosin-8-yl)-4AQO, 3-(desoxyguanosin-N 2-yl)-4AQO and 3-(deoxyadenosin-N 6-yl)-4AQO. This study was designed to assess the shape of the dose–response curve at low concentrations of 4NQO in three human lymphoblastoid cell lines, MCL-5, AHH-1 and TK6 as well as the mouse lymphoma L5178Y cell line in vitro. Chromosomal damage was investigated using the in vitro micronucleus assay, while further gene mutation and DNA damage studies were carried out using the hypoxanthine–guanine phosphoribosyltransferase forward mutation and comet assays. 4NQO showed little to no significant increases in micronucleus induction in the human lymphoblastoid cell lines, even up to 55±5% toxicity. A dose–response relationship could only be observed in the mouse lymphoma cell line L5178Y after 4NQO treatment, even at concentrations with no reduction in cell viability. Further significant increases in gene mutation and DNA damage induction were observed. Hence, 4NQO is a more effective point mutagen than clastogen, and its suitability as a positive control for genotoxicity testing has to be evaluated for every individual assay.

Relationships between p53 status, apoptosis and induction of micronuclei in different human and mouse cell lines in vitro: Implications for improving existing assays

Accumulated evidence has shown that in vitro mammalian cell genotoxicity assays produce high frequencies of “misleading” positive results, i.e. predicted hazard is not confirmed in in vivo and/or carcinogenicity studies [1], raising the question of relevance to human risk assessment. A recent study of micronucleus (MN) induction [2] showed that commonly used p53-deficient rodent cell lines (CHL, CHO and V79) gave a higher frequency of “misleading” positive results with 9 non-DNA reactive, Ames-negative and in vivo negative chemicals [3] than human p53-competent cells (blood lymphocytes, TK6 and HepG2 cell lines). This raised the question of whether these differences were due to p53 status or species origin.This present study compared human versus mouse and p53-competent versus p53-mutated function. The same 9 chemicals were tested for induction of MN in mouse lymphoma L5178Y (mutated p53), human TK6 (functional p53) and WIL2-NS (TK6 related, with mutated p53) cells. Six chemicals provided clear positive increases in MN frequency in at least one cell type. L5178Y cells yielded clear positive responses with more chemicals than either TK6 or WIL2-NS, indicating origin rather than p53 functionality was most relevant. Apoptosis induction (measured via caspase-3/7) was also investigated with clear differences in the timing and extent of apoptosis induction between mouse and human cells noted. With curcumin in TK6 cells, induction of caspase-3/7 activity coincided with MN induction, whereas for L5178Y cells, MN induction occurred in the absence of increased caspase activity. By contrast, with MMS in TK6 cells, MN induction preceded increased caspase-3/7 activity. These data suggest that MN induction by “misleading positive” genotoxins in p53-competent human cell lines may result from apoptosis, whereas in p53-defective rodent cells such as L5178Y, MN induction may be independent of apoptosis.