Mouse Lymphoma Thymidine Kinase Gene Mutation Assay: International Workshop on Genotoxicity Tests Workgroup Report—Plymouth, UK 2002

Mittelstaedt

et al. 2013

Toxicology in Vitro

Abstract2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) is a low molecular weight nitroxide and stable free radical. In this study, we investigated the cytotoxicity and genotoxicity of TEMPO in mammalian cells using the mouse lymphoma assay (MLA) and in vitro micronucleus assay. In the absence of metabolic activation (S9), 3 mM TEMPO produced significant cytotoxicity and marginal mutagenicity in the MLA; in the presence of S9, treatment of mouse lymphoma cells with 1-2 mM TEMPO resulted in dose-dependent decreases of the relative total growth and increases in mutant frequency. Treatment of TK6 human lymphoblastoid cells with 0.9-2.3 mM TEMPO increased the frequency of both micronuclei (a marker for clastogenicity) and hypodiploid nuclei (a marker of aneugenicity) in a dose-dependent manner; greater responses were produced in the presence of S9. Within the dose range tested, TEMPO induced reactive oxygen species and decreased glutathione levels in mouse lymphoma cells. In addition, the majority of TEMPO-induced mutants had loss of heterozygosity at the Tk locus, with allele loss of ≤34 Mbp.These results indicate that TEMPO is mutagenic in the MLA and induces micronuclei and hypodiploid nuclei in TK6 cells. Oxidative stress may account for part of the genotoxicity induced by TEMPO in both cell lines.

Section: Discussionmentioning

confidence: 99%

Nitroxide TEMPO: A genotoxic and oxidative stress inducer in cultured cells

Mittelstaedt

et al. 2013

Toxicology in Vitro

“…The data evaluation criteria developed by the MLA Expert Workgroup of the International Workgroup for Genotoxicity Tests (IWGT) were used for data analysis (Moore et al, 2003). For the microwell version of the MLA, a chemical is judged to be positive if there is a dose-related increase in MF and if one or more cultures show an induced mutant frequency that exceeds the global evaluation factor of 126 ϫ 10…”

Section: Identification and Quantitation Of Products Of 4-hne(ac)mentioning

confidence: 99%

“…We selected the MLA because of its ability to detect a broad spectrum of genetic damage including both point and chromosomal mutations (Hozier et al, 1981;Moore et al, 1985aMoore et al, ,b, 2002Moore et al, , 2003Applegate et al, 1990;Chen et al, 2002a,b).…”

mentioning

confidence: 99%

Mutagenic Effects of 4-Hydroxynonenal Triacetate, a Chemically Protected Form of the Lipid Peroxidation Product 4-Hydroxynonenal, as Assayed in L5178Y/Tk⁺/– Mouse Lymphoma Cells

Singh¹,

Chen²,

Chen³

et al. 2005

J Pharmacol Exp Ther

The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) is cytotoxic and genotoxic at superphysiological concentrations. To characterize the mechanism of action of 4-HNE, we assessed genotoxic damage by 4-HNE and by 4-HNE triacetate [4-HNE(Ac) 3 ] using the mouse lymphoma assay that measures the mutant frequency in the Tk gene. As a strong electrophile, 4-HNE reacts readily with nucleophilic centers on cellular components. When added extracellularly, it may react preferentially with proteins in culture medium or on the cell surface and not reach deeper cellular targets such as nuclear DNA. Therefore, 4-HNE(Ac) 3 , a protected form of 4-HNE that is metabolically converted to 4-HNE in cells (Neely MD, Amarnath V, Weitlauf C, and Montine TJ, Chem Res Toxicol 15: 40 -47, 2002), was assayed in addition to 4-HNE. When added in serum-containing medium, 4-HNE was not mutagenic in the mouse lymphoma assay up to 38 M (cytotoxicity ϭ 13%). In contrast, exposure to 4-HNE(Ac) 3 , which mimics intracellular formation of 4-HNE, resulted in dose-dependent induction of mutations. At 17 M 4-HNE(Ac) 3 (cytotoxicity ϭ 33%), the mutant frequency was 719 ϫ 10 Ϫ6 (Ͼ7-fold higher than the spontaneous mutant frequency). Loss of heterozygosity analysis in the Tk mutants revealed that the majority of mutations induced by 4-HNE(Ac) 3 resulted from clastogenic events affecting a large segment of the chromosome. The results indicate that, in the presence of serum that approximates physiological conditions, 4-HNE generated intracellularly but not extracellularly is a strong mutagen via a clastogenic action at concentrations that may occur during oxidative stress.The production of partially reduced forms of dioxygen such as superoxide, hydrogen peroxide, or the hydroxyl radical (reactive oxygen species or ROS) is an inevitable consequence of aerobic life. Because of their high reactivity, ROS and/or their downstream products such as peroxynitrite (Crow, 2000) can attack all cellular constituents. It is often tacitly assumed that functionally relevant effects of ROS are due to damage to proteins and nucleic acids because such damage results in immediate functional consequences or mutations. However, ROS also target lipids. The hydroxyl radical can initiate a chain reaction that produces multiple lipid hydroperoxide molecules from a single initial event (Gutteridge and Halliwell, 1990). Since lipid hydroperoxides are oxidants capable of damaging cellular constituents, the chain reaction is in effect amplifying the initial oxidative insult. Peroxida-

“…The results are shown in table 2. For this assay, a positive result is described as a MF that is higher than the sum of Mean Control MF (77 x 10 -6 ) plus the standard global evaluation factor for this microplate assay (GEF, 126 x 10 -6 , Moore et al 13 ). In the presence of S9, there was a slight increase in mutant frequency with the two highest doses, but the mutant frequency remained within the GEF and is therefore not considered to be a mutagenic response.…”

Section: Mouse Lymphoma Thymidine Kinase (Tk-/-) Gene Mutation Assaymentioning

confidence: 99%

The cellular toxicology of mitragynine, the dominant alkaloid of the narcotic-like herb, Mitragyna speciosa Korth

et al. 2015

Toxicology ResearchDear Editor, Thank you for the reviews on our submission. The reviewers make some very good suggestions that we have incorporated into the text and the revised manuscript is now much stronger. I shall address the reviewers' comments on a point by point basis. Referee 1This is a well written paper and the experiments described, though fairly basic, are well performed and analysed. The work explores the mechanism of toxicity of mitragynine, the active alkaloid constituent of the herb Mitragyna speciosa Korth, and suggests a key molecular initiating event (MIE) that triggers the toxicity cascade.We thank the reviewer for the supportive words. The dose level at which toxicity was seen is 75µM that is some 75,000 to 750fold higher that that found in fairly high level users of the herb. This needs to be included in the abstract.We do mention this fact in the discussion along with the problems of extrapolating in vitro data to the situation in humans. To put our data in context, we also comment upon the human fatalities associated with Kratom consumption. In line with the comment and taking into consideration the reviewer's point 2, we now include new statements in the abstract and expand on this in the discussion. This is a great point. In fact references 9-11 in our manuscript give details of plasma levels of mitragynine in human fatalities associated with Kratom use. The plasma mitragynine concentrations in these individuals varied between 0.45 -1.5 microM, which is 50 fold less than the concentrations used in our studies. We have therefore included a review of these reports in our revised discussion. It Shown in both the cytoxicity assays (fig 2B) and clonogenic assays (Fig 2D) the lower doses show a marked cell proliferation. This is not commented on and some text should be included to deal with this.We have amended the text to comment on this. The reviewer makes an important point and we have reviewed the literature around this. We note that there is clear evidence in the literature of opiate mediated cell toxicity, particularly around morphine. In the revised submission we now reference reports of similar observations to ours, regarding naloxone effects on morphine toxicity and their speculation on the role of the µ receptor. Clearly the role of opioid receptors in opiate cellular toxicity remains controversial. The authors looked at the mechanism and hypothesised that the key molecular event could be interaction with the opiate receptors. This was tested using antagonists against these receptors. Naloxone that has activity particularly against the µ and ҝ receptors showed activity in reducing the toxicity but naltrindole (active against the δ receptor) did not show activity in Minor comments a. The resolution of figure 1 needs to be improved. This figure has clearly been cut and pasted from elsewhere.We have redrawn the figure using Chem-Draw. b. Figures 2A and 2B require error bars.We apologise for this oversight. The error bars have been added. Referee: 2Comments to the Author The results f...