New Acyclic Cytotoxic Jasplakinolide Derivative from the Marine Sponge Jaspis splendens.

A new acylic jasplakinolide congener (2), another acyclic derivative requiring revision (4), together with two jasplakinolide derivatives including the parent compound jasplakinolide (1) were isolated from the Indonesian marine sponge Jaspis splendens. The chemical structures of the new and known compounds were unambiguously elucidated based on HRESIMS and exhaustive 1D and 2D NMR spectral analysis as well as a comparison of their NMR data with those of jasplakinolide (1). The isolated jasplakinolides inhibited the growth of mouse lymphoma (L5178Y) cells in vitro with IC50 values in the low micromolar to nanomolar range.

Whole genome sequencing analysis of small and large colony mutants from the mouse lymphoma assay.

The Thymidine kinase (Tk) gene forward mutation assay, known as the mouse lymphoma assay (MLA), has been widely used for evaluating the genotoxicity of chemical agents. A striking morphological feature of Tk mutant colonies is the bimodal distribution of their sizes, with cells from the large colonies growing at a normal rate and cells from the small colonies growing at a slower rate than normal. To understand the molecular distinction for the different growth rates, we performed whole genome sequencing (WGS) analysis of the large and small colony mutants generated from the MLA. Three large colony and three small colony mutants generated from cells treated with 4-nitroquinoline 1-oxide (4-NQO) or the vehicle control were selected for analysis. The WGS data were analyzed for loss of heterozygosity (LOH) and chromosome copy number along chromosome 11, where the Tk gene is located. Although there were LOH alterations in both large and small colony mutants, copy number changes near Tk locus were found only in small colony mutants produced by the vehicle control and 4-NQO treatments. The chromosome copy number in the regions near the Tk locus increased from two to three or four in the spontaneous small colony mutants and decreased from two to one in the 4-NQO-induced small colony mutants. These results suggest that chromosome damage was repaired differently in the large and small colony mutants, resulting in significant chromosome alterations in the small colony mutants, but not in the large colony mutants. Thus, chromosome alterations near the Tk locus may play a major role in the inhibition of cell growth in the Tk small colony mutants.

Uptake and metabolism of mizoribine, an immunosuppressant, in L5178Y-R mouse lymphoma cells in vitro and peripheral blood mononuclear cells of rats and kidney transplant recipients in vivo.

The cellular uptake of mizoribine (MZR), an immunosuppressant, and metabolism of MZR to MZR-5'- monophosphate (MZRP), an active metabolite, were evaluated in L5178Y-R mouse lymphoma cells and peripheral blood mononuclear cells (PBMCs) of rats and kidney transplant recipients (KTRs, n = 22). Real-time PCR analysis revealed the expression of ENT1 and ENT2 mRNAs, but not of CNTs, in L5178Y-R cells and rat's PBMCs. In L5178Y-R cells, the uptake of MZR was suppressed by adenosine, a substrate for ENT1 and ENT2, but not by 5-(4-nitrobenzyl)-6-thioinosine (0.1 µM), an ENT1 inhibitor. Saturable metabolism of MZR to MZRP was observed. In rats, peak plasma concentrations of MZR and peak concentrations of MZR and MZRP in PBMCs were observed 3 h after oral administration. MZR disappeared from PBMCs in parallel with plasma MZR, but the disappearance of MZRP from PBMCs appeared to be slow. In KTRs, the mean plasma concentration of MZR 3-4 h after ingestion was 3.14 µg/ml and the mean MZRP concentration in PBMCs was 16.8% of MZR, reflecting the involvement of ENT in the uptake of MZR. A linear relationship was observed between plasma MZR concentrations ranging from 1 to 6 µg/ml and PBMC's MZRP concentrations ranging from 90 to 200 ng/ml.

Lactones from the Sponge-Derived Fungus Talaromyces rugulosus.

The marine-derived fungus Talaromyces rugulosus isolated from the Mediterranean sponge Axinella cannabina and cultured on solid rice medium yielded seventeen lactone derivatives including five butenolides (1-5), seven (3S)-resorcylide derivatives (6-12), two butenolide-resorcylide dimers (13 and 14), and three dihydroisocoumarins (15-17). Among them, fourteen compounds (1-3, 6-16) are new natural products. The structures of the isolated compounds were elucidated by 1D and 2D NMR (Nuclear Magnetic Resonance) spectroscopy as well as by ESI-HRMS (ElectroSpray Ionization-High Resolution Mass Spectrometry). TDDFT-ECD (Time-Dependent Density Functional Theory-Electronic Circular Dichroism) calculations were performed to determine the absolute configurations of chiral compounds. The butenolide-resorcylide dimers talarodilactones A and B (13 and 14) exhibited potent cytotoxicity against the L5178Y murine lymphoma cell line with IC50 values of 3.9 and 1.3 µM, respectively.

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.

The Modified Heparin-Binding L-Asparaginase of Wolinella succinogenes.

The modified asparaginase Was79 was derived from the recombinant wild-type L-asparaginase of Wolinella succinogenes. The Was79 contains the amino acid substitutions V23Q and K24T responsible for the resistance to trypsinolysis and the N-terminal heparin-binding peptide KRKKKGKGLGKKR responsible for the binding to heparin and tumor K562 cells in vitro. When tested on a mouse model of Fischer lymphadenosis L5178Y, therapeutic efficacy of Was79 was significantly higher than that of reference enzymes at all single therapeutic doses used (125-8000 IU/kg). At Was79 single doses of 500-8000 IU/kg, the complete remission rate of 100 % was observed. The Was79 variant can be expressed intracellularly in E. coli as a less immunogenic formyl-methionine-free form at high per cell production levels.

In vitro genotoxicity testing of carvacrol and thymol using the micronucleus and mouse lymphoma assays.

Currently, antimicrobial additives derived from essential oils (Eos) extracted from plants or spices, such as Origanum vulgare, are used in food packaging. Thymol and carvacrol, the major EO compounds of O. vulgare, have demonstrated their potential use as active additives. These new applications use high concentrations, thereby increasing the concern regarding their toxicological profile and especially their genotoxic risk. The aim of this work was to investigate the potential in vitro genotoxicity of thymol (0-250 µM) and carvacrol (0-2500 µM) at equivalent doses to those used in food packaging. The micronucleus (MN) test and the mouse lymphoma (MLA) assay on L5178Y/Tk(±) mouse lymphoma cells were used. The negative results for thymol with the MN with and without the S9 fraction and also with the MLA assay reinforce the view that this compound is not genotoxic in mammalian cells. However, carvacrol presented slight genotoxic effects, but only in the MN test at the highest concentration assayed (700 µM) and in the absence of metabolic activation. The lack of genotoxic response in the MLA assay after 4 and 24h of exposure indicates a low genotoxic potential for carvacrol. Alternatively, the general negative findings observed in both assays suggest that the MN results of carvacrol are marginal data without biological relevance. These results can be useful to identify the appropriate concentrations of these substances to be used as additives in food packaging.

Preclinical studies on the pharmacokinetics, safety, and toxicology of oxfendazole: toward first in human studies.

A 2-week study in rats identified target organs of oxfendazole toxicity to be bone marrow, epididymis, liver, spleen, testis, and thymus. Female rats had greater oxfendazole exposure and exhibited toxicities at lower doses than did males. Decreased white blood cell levels, a class effect of benzimidazole anthelmintics, returned to normal during the recovery period. The no observed adverse effect level was determined to be >5 but <25 mg/kg/d and the maximum tolerated dose 100 mg/kg/d. The highest dose, 200 mg/kg/d, resulted in significant toxicity and mortality, leading to euthanization of the main study animals in this group after 7 days. Oxfendazole did not exhibit genetic toxicology signals in standard Ames bacterial, mouse lymphoma, or rat micronucleus assays nor did it provoke safety concerns when evaluated for behavioral effects in rats or cardiovascular safety effects in dogs. These results support the transition of oxfendazole to First in Human safety studies preliminary to its evaluation in human helminth diseases.

From radioresistance to radiosensitivity: In vitro evolution of L5178Y lymphoma.

PURPOSE: To discuss the possible reasons for the loss of tumourigenicity and the acquisition of new phenotypic features (among them, sensitivity to X and UVC radiations) as a result of in vitro cultivation of L5178Y lymphoma cells. RESULTS: Ten years ago the phenotypic differences between LY-R (original L5178Y maintained in vivo and examined in vitro) and LY-S lines were reviewed in detail by the author. The loss of tumourigenicity of LY-R cells upon in vitro cultivation accompanying the acquirement of the LY-S phenotype had been described earlier by Beer et al. (1983). In spite of their common origin, the sublines were shown to differ in their relative sensitivity to a number of DNA damaging agents and in numerous other features. Here, selected differences between LY-R and LY-S lines are briefly reviewed. It is proposed that Wallace's concept (2010a) that mitochondria are the interface between environmental conditions and the genome may explain the LY-R-LY-S conversion under prolonged in vitro cultivation. CONCLUSION: The differences between the LY lines were probably of epigenetic rather than genetic character. The properties of LY-R cells changed as a result of exposure to an oxic in vitro milieu. The changes could be preconditioned by heteroplasmy and the selection of cells endowed with mitochondria best fitted to a high oxygen-low carbon dioxide environment.

Can the L5178Y Tk+/- mouse lymphoma assay detect epigenetic silencing?

The mouse lymphoma L5178Y Tk(+/-) assay is broadly used in toxicology to assess genotoxicity because of its known sensitivity to genotoxicants that act through a variety of mechanisms, which may include epigenetic DNA methylation. This brief article highlights the studies that have contributed to this conjecture and suggests an addition to the experimental design that could identify if the test substance is a potential epimutagen acting via hypermethylation.

Effect of ß-carotene on catechol-induced genotoxicity in vitro: evidence of both enhanced and reduced DNA damage.

Intake of antioxidants from the diet has been recognized to have beneficial health effects, but the potential benefit of taking antioxidants such as ß-carotene as supplements is controversial. The aim of the present study was to evaluate the potential protective effects of a physiologically relevant concentration (2 µM) of ß-carotene on the DNA damaging effects of catechol in mouse lymphoma L5178Y cells. Two different exposure protocols were used: simultaneous exposure to ß-carotene and catechol for 3 h; and exposure to catechol for 3 h after 18 h pre-treatment with the vitamin. DNA damage was evaluated using the comet assay (employing one procedure for general damage, and another procedure, which also included oxidative DNA damage). Independent of exposure protocol and procedure for comet assay, ß-carotene did not increase the basal level of DNA damage. However, at the highest concentration of catechol (1 mM), ß-carotene was found to clearly increase the level of catechol-induced DNA damage, especially in the pre-treated cells. Interestingly, an opposite effect was observed at lower concentrations of catechol, but the ß-carotene related reduction of catechol-induced genotoxicity was significant (P < 0.05) only for the procedure including oxidative damage induced by 0.5 mM catechol. Taken together our results indicate that ß- carotene can both reduce and enhance the DNA damaging effects of a genotoxic agent such as catechol. This indicates that it is the level of catechol-induced DNA damage that seems to determine whether ß-carotene should be regarded as a beneficial or detrimental agent when it comes to its use as a dietary supplement.

The effect of long term storage on tobacco smoke particulate matter in in vitro genotoxicity and cytotoxicity assays.

Particulate matter (PM) collected from mainstream tobacco smoke is a test article commonly used for in vitro genotoxicity and cytotoxicity testing of combustible tobacco products. However, little published data exists concerning the stability of PM. We completed a 2 year study to quantify the effect of PM storage at -80 °C, on the genotoxicity and cytotoxicity of PM generated from 3R4F and M4A reference cigarettes. The Ames test, Micronucleus assay (MNvit), Mouse Lymphoma assay (MLA) and the Neutral Red Uptake assay (NRU) were used. The majority of M4A and 3R4F PMs were genotoxic and cytotoxic at the timepoints tested. Some minor but statistically significant differences were observed for stored versus freshly prepared PM, but the magnitude of changes were within the variability observed for repeat testing.

In vitro cytotoxic activity of novel protoflavone analogs - selectivity towards a multidrug resistant cancer cell line.

BACKGROUND: Protoapigenone (PA), a natural flavonoid possessing an unusual p-quinol moiety on its B ring, is a prospective novel lead compound against cancer currently in development, together with WYC0209, a potent synthetic PA analog. Structure activity relationships (SAR) concerning different 1'-O-alkyl side-chains were also studied on two sets of derivatives. MATERIALS AND METHODS: Fifteen 1'-O-alkyl protoflavone derivatives were synthesized from genkwanin or 4'-hydroxy-6-methylflavone, thirteen of which are new compounds. All compounds were tested for their cytotoxic effect on four human cancer cell lines, such as HepG2 and Hep3B (hepatic), A549 (lung) and MDA-MB-231 (breast) cell lines, with doxorubicin as a positive control. All compounds, as well as PA, WYC0209 and fourteen of their previously reported analogs were also tested on a multidrug-resistant (MDR) sub-cell line of L5178 mouse T-cell lymphoma and on its parental counterpart (PAR). RESULTS: In general, derivatives bearing a free hydroxyl group at C-1' exerted the strongest activities, while C-1'-substituted compounds were found to be much weaker. Derivatives of 6-methylflavone exhibited mild, but statistically significant selectivity towards the MDR cell line. CONCLUSION: The results are in agreement with our previous findings for fundamental SAR of protoflavones. 6-Methylated protoflavones may serve as valuable leads for developing selective compounds against MDR cancer. Identical activity of other derivatives on the PAR and MDR cell lines suggests that cancer cells cannot exhibit resistance to protoflavones by ABCB1 efflux pump overexpression.

Growth retardation of Paramecium and mouse cells by shielding them from background radiation.

In the 1970s and 1980s, Planel et al. reported that the growth of paramecia was decreased by shielding them from background radiation. In the 1990s, Takizawa et al. found that mouse cells displayed a decreased growth rate under shielded conditions. The purpose of the present study was to confirm that growth is impaired in organisms that have been shielded from background radiation. Radioprotection was produced with a shielding chamber surrounded by a 15 cm thick iron wall and a 10 cm thick paraffin wall that reduced the γ ray and neutron levels in the chamber to 2% and 25% of the background levels, respectively. Although the growth of Paramecium tetraurelia was not impaired by short-term radioprotection (around 10 days), which disagreed with the findings of Planel et al., decreased growth was observed after long-term (40-50 days) radiation shielding. When mouse lymphoma L5178Y cells were incubated inside or outside of the shielding chamber for 7 days, the number of cells present on the 6th and 7th days under the shielding conditions was significantly lower than that present under the non-shielding conditions. These inhibitory effects on cell growth were abrogated by the addition of a ¹³7Cs γ-ray source disk to the chamber. Furthermore, no growth retardation was observed in XRCC4-deficient mouse M10 cells, which display impaired DNA double strand break repair.

Genetic damage, but limited evidence of oxidative stress markers in diethyl maleate-induced glutathione depleted mouse lymphoma L5178Y (TK(+/-)) cell cultures.

Depletion of glutathione (GSH) in cells exposed to certain xenobiotics has been proposed to result in oxidative stress, which could lead to damage of cellular macromolecules such as proteins, lipids, and DNA. Diethyl maleate (DEM) is known to conjugate with GSH and rapidly lower cellular GSH levels. The objective of this study was to investigate the influence of DEM-induced GSH depletion on various genotoxicity and gene expression end points in mouse lymphoma L5178Y (TK(+/-)) cell cultures. Cells were exposed to DEM for 4 h at concentrations of 0, 6.7, 13.5, 26.9, 53.8, 107.6, 215.3, and 430.6 µg/mL (0.039-2.5 mM). Genotoxicity was evaluated by examining the induction of in vitro micronuclei (20 h post-treatment) and DNA strand breaks as measured by comet (immediately following treatment), and correlating these observations to cellular GSH levels. In the current study, GSH was decreased more than 50% at the lowest test concentration (6.7 µg/mL) and more than 95% at ≥ 107.6 µg/mL. A significant increase in micronuclei and DNA strand breaks was observed at concentrations of ≥ 26.9 µg/mL. Gene expression of seven apoptosis and oxidative-stress related genes showed significant alterations in only three genes only at the highest test concentration. Quantifiable levels of 8-OH-dG (≥ 2 adducts per 1 × 10(8) NT) were not detected at any treatment concentration. These results demonstrate an association between DEM-induced genotoxicity and GSH depletion in mouse lymphoma L5178Y (TK(+/-)) cells, but not with other oxidative markers.

Activity of fourteen new hydantoin compounds on the human ABCB1 efflux pump.

BACKGROUND: Multidrug resistance (MDR) is one of the major concerns in the treatment of cancer and one of the major causes of therapy failure. The overexpression of an ABC transporter, the ABCB1, is often associated with MDR in cancer. Previously it was observed that hydantoin compounds can modulate the activity of the ABCB1 pump. MATERIALS AND METHODS: Fourteen hydantoin derivatives were synthesized and studied for their capacity to increase accumulation of ethidium bromide (EB) by mouse lymphoma cancer cells that were transfected with the human ABCB1 gene and overexpress the human ABCB1 pump. RESULTS: It was observed that the accumulation of EB by the cells in the presence of four of the newly synthesized hydantoins was strongly increased. Similar but milder effects were also observed for the other seven hydantoins; the remaining three had no activity. CONCLUSION: The 14 hydantoin compounds studied belong to three different structural groups. Structure-activity relationships were studied and important molecular substituents that were possibly responsible for increased the activity of the molecules were identified. This important information may lead to the continuation of our work and to the future synthesis of more active compounds.

Genotoxicity testing of a Hoodia gordonii extract.

Hoodia gordonii extract consists of a mixture of steroid glycosides, fatty acids, plant sterols and alcohols. As part of the overall safety assessment H. gordonii extract was assessed for genotoxicity in two assays in vitro: a bacterial mutation assay; and a gene mutation assay using mouse lymphoma cells. H. gordonii extract showed no evidence of genotoxic activity in either of these assays. In addition, H. gordonii extract was assessed for mutagenic activity in a bone marrow micronucleus (MN) assay in the mouse, with 400mg/kg selected as the high-dose group, based on observations in a dose-range-finding study. The group mean frequencies of micronucleated polychromatic erythrocytes of treated animals were similar to those of the vehicle control group, indicating H. gordonii extract to be non-genotoxic under the conditions of this test. All assays were performed in compliance with the Good Laboratory Practice Regulations and in accordance with standard guidelines for genotoxicity tests. H. gordonii extract was shown to be non-genotoxic in 3 independent assays (a bacterial mutation test, a gene mutation assay using mouse lymphoma cells and a bone marrow micronucleus assay in the mouse).

The mouse lymphoma assay.

The mouse lymphoma TK assay (MLA) is part of an in vitro battery of tests designed to predict risk assessment prior to in vivo testing. The test has the potential to detect mutagenic and clastogenic events at the thymidine kinase (tk) locus of L5178Y mouse lymphoma tk ( +/- ) cells by measuring resistance to the lethal nucleoside analogue triflurothymidine (TFT). Cells may be plated for viability and mutation in semi-solid agar (agar assay) or in 96-well microtitre plates (microwell assay). When added to selective medium containing TFT, wild-type tk ( +/- ) cells die, but TFT cannot be incorporated into the DNA of mutant tk ( -/- ) cells, which survive to form colonies that may be large (indicative of gene mutation) or small (indicative of chromosomal mutation) in nature. Mutant frequency is expressed as the number of mutants per 10(6) viable cells.

A rapid, semi-automated method for scoring micronuclei in mononucleated mouse lymphoma cells.

A semi-automated scoring system has been developed to provide rapid, accurate assessment of micronuclei in preparations of mononuclear mouse lymphoma L5178Y cells. Following exposure to a range of test agents, flat, single-cell preparations were produced from exponentially growing cultures by cytocentrifugation. Following staining with 4'-6-diamidino-2-phenylindole (DAPI), cells were scanned by use of the MicroNuc module of Metafer 4 v 3.4.102, after modifying the classifier developed for selecting micronuclei in binucleate cells to increase its sensitivity. The image gallery of all cells was then sorted to bring aberrant cells to the top of the gallery to assess visually the numbers of cells with micronuclei, as distinct from other debris. Slide quality was shown to be paramount in obtaining accurate results from an automated scan and the data obtained compared very well with the incidence of micronuclei scored conventionally by microscopy. Compared with manual scoring the time saving is considerable, as more than 2000 images are captured in approximately 2min, with subsequent visual assessment of aberrant cells in the image gallery taking about 1-2min/slide. By scanning all aberrant cells, the system also captures additional information on necrotic, apoptotic and fragmented cells. Although optimised for mouse lymphoma cells, it should be simple to adapt the method for any cell type growing in suspension.

Genotoxicity screening via the γH2AX by flow assay.

The measurement of serine139-phosphorylated histone H2AX (γH2AX) provides a biomarker of DNA double-strand breaks (DSBs) and may identify potential genotoxic activity. In order to evaluate a flow cytometry assay for γH2AX detection (hereafter termed the γH2AX by flow assay), 6 prototypical (3 pro- and 3 proximate) genotoxins, i.e. dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P), methyl methane sulphonate (MMS), methyl nitrosourea (MNU) and 4-nitroquinoline oxide (4NQO), were selected to define assay evaluation criteria. In addition, 3 non-genotoxic cytotoxins (phthalic anhydride, n-butyl chloride and hexachloroethane) were included to investigate the influence of cytotoxicity on assay performance. At similar cytotoxicity levels (relative cell counts; RCC 75-40%) all prototypical genotoxins induced marked concentration-dependent increases in γH2AX compared with the non-genotoxins. As a result, assay evaluation criteria for a positive effect were defined as >1.5-fold γH2AX @ RCC >25%. Twenty five additional chemicals with diverse structures and genotoxic activity were selected to evaluate the γH2AX by flow assay. Results were compared with Ames bacterial and in vitro mammalian genotoxicity tests (mouse lymphoma assay and/or chromosome aberration assay). γH2AX by flow assay results were highly predictive of Ames (sensitivity 100%; specificity 67%; concordance 82%) and in vitro mammalian genotoxicity tests (sensitivity 91%; specificity 89%; concordance 91%) and provide additional evidence that γH2AX is a biomarker of potential genotoxic activity, underpinned mechanistically by the cellular response to DSBs. Discordant findings were predominately attributed to differences in specificity for some mammalian cell genotoxins that are Ames non-mutagens or for "biologically-irrelevant" positives in the mammalian tests. Simple anilines were classified as genotoxic following rat liver S9-mediated bioactivation, however, effects on γH2AX were atypical and limited to a small sub-population of S-phase nuclei. Nevertheless, the γH2AX by flow assay represents a novel genotoxicity assay with the potential to flag both pro- and proximate genotoxins.