Comparative Genotoxicity of TEMPO and 3 of Its Derivatives in Mouse Lymphoma Cells.

TEMPO (2, 2, 6, 6-tetramethylphiperidine-1-oxyl) and its derivatives are stable free radical nitroxides widely used in the field of chemistry, biology, and pharmacology. TEMPO was previously found to be mutagenic and to induce micronuclei in mammalian cells. In this study, we investigated and quantified the genotoxicity of 4 structurally similar nitroxides, TEMPO and 3 of its derivatives (4-hydroxy-TEMPO, 4-oxo-TEMPO, and 4-methoxy-TEMPO), using the mouse lymphoma assay (MLA) and Comet assay in L5178Y Tk+/- cells. The results showed that all tested nitroxides were cytotoxic and mutagenic in the MLA, both in the presence and absence of S9, with metabolic activation significantly enhancing the cytotoxicity and/or mutagenicity. In addition, the 4 nitroxides caused DNA-strand breakage. The mutagenicity and DNA damaging dose-responses of the test articles were compared using the PROAST benchmark dose software package. The potency ranking of the 4 nitroxides for mutagenicity was different from the ranking of the DNA damaging effects. The mode of action analysis by a multi-endpoint DNA damage pathway assay classified all 4 nitroxides as clastogens. In addition, the majority of the induced Tk mutants showed loss of heterozygosity at the Tk and D11Mit42 loci (ie, chromosome damage <31 Mbp). These results suggest that TEMPO and its 3 derivatives are cytotoxic and mutagenic in mouse lymphoma cells through a mechanism that involves strand breakage and large alterations to DNA. The potency rankings indicate that the different TEMPO derivatives vary in their mutagenic and DNA damaging potential.

Quantitative differentiation of whole smoke solution-induced mutagenicity in the mouse lymphoma assay.

In vitro genotoxicity dose-response data have been investigated for their utility in modeling and assessing potential differences in mutagenic responses between machine-generated whole smoke solutions (WSSs) from combusted cigarette tobacco products. Our previous study observed that potency ranking by benchmark dose (BMD) analysis was a useful modeling approach for quantitative assessment of differences between the mutagenicity of several structurally diverse chemical constituents of cigarette smoke. To follow-up on these observations, we used the mouse lymphoma assay (MLA) to evaluate the mutagenicity of WSSs prepared from two commercial cigarettes smoked under two different smoking machine regimens. L5178Y cells were exposed to ≥5 concentrations of each WSS for 4 hr ± S9 activation. S9 reduced the cytotoxicity and enhanced the mutagenicity of the WSSs. The resulting S9-mediated mutagenicity dose-responses were compared between test articles using BMD analysis, the lowest dose exceeding the Global Evaluation Factor, the no observed or lowest observed genotoxic effect level, and the mutagenic potency. The BMD10 , BMD50 , BMD100 , and BMD200 , indicating a 10%, 50%, 100%, or 200% increase in the background mutant frequency, respectively, were calculated using the PROAST software package. Overall, the quantitative approaches resulted in a similar rank order of mutagenic potency for the MLA tested WSSs, with potency increasing with the level of tar. The BMD approach using covariate analysis produced the most informative comparisons. Differences in potency were associated with the number of cigarettes smoked, the cigarette product smoked, and the smoking machine protocol used to prepare the sample. Under the conditions of this study, these results suggest that our hypothesis of modeling MLA data using the BMD approach to quantitatively discriminate between the mutagenic potential of WSSs from combustible cigarettes might be an useful method. Environ. Mol. Mutagen. 59:103-113, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA.

Quantitative analysis of the relative mutagenicity of five chemical constituents of tobacco smoke in the mouse lymphoma assay.

Quantifying health-related biological effects, like genotoxicity, could provide a way of distinguishing between tobacco products. In order to develop tools for using genotoxicty data to quantitatively evaluate the risk of tobacco products, we tested five carcinogens found in cigarette smoke, 4-aminobiphenyl (4-ABP), benzo[a]pyrene (BaP), cadmium (in the form of CdCl2), 2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (MeIQ) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in the mouse lymphoma assay (MLA). The resulting mutagenicity dose responses were analyzed by various quantitative approaches and their strengths and weaknesses for distinguishing responses in the MLA were evaluated. L5178Y/Tk (+/-) 3.7.2C mouse lymphoma cells were treated with four to seven concentrations of each chemical for 4h. Only CdCl2 produced a positive response without metabolic activation (S9); all five chemicals produced dose-dependent increases in cytotoxicity and mutagenicity with S9. The lowest dose exceeding the global evaluation factor, the benchmark dose producing a 10%, 50%, 100% or 200% increase in the background frequency (BMD10, BMD50, BMD100 and BMD200), the no observed genotoxic effect level (NOGEL), the lowest observed genotoxic effect level (LOGEL) and the mutagenic potency expressed as a mutant frequency per micromole of chemical, were calculated for all the positive responses. All the quantitative metrics had similar rank orders for the agents' ability to induce mutation, from the most to least potent as CdCl2(-S9) > BaP(+S9) > CdCl2(+S9) > MeIQ(+S9) > 4-ABP(+S9) > NNK(+S9). However, the metric values for the different chemical responses (i.e. the ratio of the greatest value to the least value) for the different chemicals ranged from 16-fold (BMD10) to 572-fold (mutagenic potency). These results suggest that data from the MLA are capable of discriminating the mutagenicity of various constituents of cigarette smoke, and that quantitative analyses are available that can be useful in distinguishing between the exposure responses.

Reactive oxygen species and c-Jun N-terminal kinases contribute to TEMPO-induced apoptosis in L5178Y cells.

The biological consequences of exposure to piperidine nitroxides is a concern, given their widespread use in manufacturing processes and their potential use in clinical applications. Our previous study reported that TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl), a low molecular weight free radical, possesses pro-oxidative activity in L5178Y cells. In this study, we investigated and characterized the role of reactive oxygen species (ROS) in TEMPO-induced toxicity in L5178Y cells. We found that TEMPO induced time- and concentration-dependent intracellular ROS production and glutathione depletion. TEMPO also induced apoptosis as demonstrated by increased caspase-3/7 activity, an increased proportion of annexin V stained cells, and decreased expression of anti-apoptotic proteins including Bcl-2, Bcl-xL and Mcl-1. N-acetylcysteine, a ROS scavenger, attenuated the ROS production and apoptosis induced by TEMPO. Moreover, Western blot analyses revealed that TEMPO activated γ-H2A.X, a hallmark of DNA damage, and c-Jun N-terminal kinases (JNK), a key member in the mitogen-activated protein kinase (MAPK) signaling pathway. Addition of SP600125, a JNK-specific inhibitor, blocked TEMPO-mediated JNK phosphorylation and also attenuated TEMPO-induced apoptosis. These findings indicate that both ROS production and JNK activation are involved in TEMPO-induced apoptosis, and may contribute to the toxicity of TEMPO in L5178Y cells.

Human sex hormone-binding globulin binding affinities of 125 structurally diverse chemicals and comparison with their binding to androgen receptor, estrogen receptor, and α-fetoprotein.

One endocrine disruption mechanism is through binding to nuclear receptors such as the androgen receptor (AR) and estrogen receptor (ER) in target cells. The concentration of a chemical in serum is important for its entry into the target cells to bind the receptors, which is regulated by the serum proteins. Human sex hormone-binding globulin (SHBG) is the major transport protein in serum that can bind androgens and estrogens and thus change a chemical's availability to enter the target cells. Sequestration of an androgen or estrogen in the serum can alter the chemical elicited AR- and ER-mediated responses. To better understand the chemical-induced endocrine activity, we developed a competitive binding assay using human pregnancy plasma and measured the binding to the human SHBG for 125 structurally diverse chemicals, most of which were known to bind AR and ER. Eighty seven chemicals were able to bind the human SHBG in the assay, whereas 38 chemicals were nonbinders. Binding data for human SHBG are compared with that for rat α-fetoprotein, ER and AR. Knowing the binding profiles between serum and nuclear receptors will improve assessment of a chemical's potential for endocrine disruption. The SHBG binding data reported here represent the largest data set of structurally diverse chemicals tested for human SHBG binding. Utilization of the SHBG binding data with AR and ER binding data could enable better evaluation of endocrine disrupting potential of chemicals through AR- and ER-mediated responses since sequestration in serum could be considered.

Mechanistic evaluation of Ginkgo biloba leaf extract-induced genotoxicity in L5178Y cells.

Ginkgo biloba has been used for many thousand years as a traditional herbal remedy and its extract has been consumed for many decades as a dietary supplement. Ginkgo biloba leaf extract is a complex mixture with many constituents, including flavonol glycosides and terpene lactones. The National Toxicology Program 2-year cancer bioassay found that G. biloba leaf extract targets the liver, thyroid gland, and nose of rodents; however, the mechanism of G. biloba leaf extract-associated carcinogenicity remains unclear. In the current study, the in vitro genotoxicity of G. biloba leaf extract and its eight constituents was evaluated using the mouse lymphoma assay (MLA) and Comet assay. The underlying mechanisms of G. biloba leaf extract-associated genotoxicity were explored. Ginkgo biloba leaf extract, quercetin, and kaempferol resulted in a dose-dependent increase in the mutant frequency and DNA double-strand breaks (DSBs). Western blot analysis confirmed that G. biloba leaf extract, quercetin, and kaempferol activated the DNA damage signaling pathway with increased expression of γ-H2AX and phosphorylated Chk2 and Chk1. In addition, G. biloba leaf extract produced reactive oxygen species and decreased glutathione levels in L5178Y cells. Loss of heterozygosity analysis of mutants indicated that G. biloba leaf extract, quercetin, and kaempferol treatments resulted in extensive chromosomal damage. These results indicate that G. biloba leaf extract and its two constituents, quercetin and kaempferol, are mutagenic to the mouse L5178Y cells and induce DSBs. Quercetin and kaempferol likely are major contributors to G. biloba leaf extract-induced genotoxicity.

In vitro investigation of the mutagenic potential of Aloe vera extracts.

A 2-year cancer bioassay in rodents with a preparation of Aloe vera whole leaf extract administered in drinking water showed clear evidence of carcinogenic activity. To provide insight into the identity and mechanisms associated with mutagenic components of the Aloe vera extracts, we used the mouse lymphoma assay to evaluate the mutagenicity of the Aloe vera whole leaf extract (WLE) and Aloe vera decolorized whole leaf extract (WLD). The WLD extract was obtained by subjecting WLE to activated carbon-adsorption. HPLC analysis indicated that the decolorization process removed many components from the WLE extract, including anthraquinones. Both WLE and WLD extracts showed cytotoxic and mutagenic effects in mouse lymphoma cells but in different concentration ranges, and WLD induced about 3-fold higher levels of intracellular reactive oxygen species than WLE. Molecular analysis of mutant colonies from cells treated with WLE and WLD revealed that the primary type of damage from both treatments was largely due to chromosome mutations (deletions and/or mitotic recombination). The fact that the samples were mutagenic at different concentrations suggests that while some mutagenic components of WLE were removed by activated carbon filtration, components with pro-oxidant activity and mutagenic activity remained. The results demonstrate the utility of the mouse lymphoma assay as a tool to characterize the mutagenic activity of fractionated complex botanical mixtures to identify bioactive components.

Rat α-Fetoprotein binding affinities of a large set of structurally diverse chemicals elucidated the relationships between structures and binding affinities.

Endocrine disrupting chemicals interfere with the endocrine system in animals, including humans, to exert adverse effects. One of the mechanisms of endocrine disruption is through the binding of receptors such as the estrogen receptor (ER) in target cells. The concentration of any chemical in serum is important for its entry into the target cells to bind the receptors. α-Fetoprotein (AFP) is a major transport protein in rodent serum that can bind with estrogens and thus change a chemical's availability for entrance into the target cell. Sequestration of an estrogen in the serum can alter the chemical's potential for disrupting estrogen receptor-mediated responses. To better understand endocrine disruption, we developed a competitive binding assay using rat amniotic fluid, which contains very high levels of AFP, and measured the binding to the rat AFP for 125 structurally diverse chemicals, most of which are known to bind ER. Fifty-three chemicals were able to bind the rat AFP in the assay, while 72 chemicals were determined to be nonbinders. Observations from closely examining the relationship between the binding data and structures of the tested chemicals are rationally explained in a manner consistent with proposed binding regions of rat AFP in the literature. The data reported here represent the largest data set of structurally diverse chemicals tested for rat AFP binding. The data assist in elucidating binding interactions and mechanisms between chemicals and rat AFP and, in turn, assist in the evaluation of the endocrine disrupting potential of chemicals.

Genomic analysis of microRNA time-course expression in liver of mice treated with genotoxic carcinogen N-ethyl-N-nitrosourea.

BACKGROUND: Dysregulated expression of microRNAs (miRNAs) has been previously observed in human cancer tissues and shown promise in defining tumor status. However, there is little information as to if or when expression changes of miRNAs occur in normal tissues after carcinogen exposure. RESULTS: To explore the possible time-course changes of miRNA expression induced by a carcinogen, we treated mice with one dose of 120 mg/kg N-ethyl-N-nitrosourea (ENU), a model genotoxic carcinogen, and vehicle control. The miRNA expression profiles were assessed in the mouse livers in a time-course design. miRNAs were isolated from the livers at days 1, 3, 7, 15, 30 and 120 after the treatment and their expression was determined using a miRNA PCR Array. Principal component analysis of the miRNA expression profiles showed that miRNA expression at post-treatment days (PTDs) 7 and 15 were different from those at the other time points and the control. The number of differentially expressed miRNAs (DEMs) changed over time (3, 5, 14, 32, 5 and 5 at PTDs 1, 3, 7, 15, 30 and 120, respectively). The magnitude of the expression change varied with time with the highest changes at PTDs 7 or 15 for most of the DEMs. In silico functional analysis of the DEMs at PTDs 7 and 15 indicated that the major functions of these ENU-induced DEMs were associated with DNA damage, DNA repair, apoptosis and other processes related to carcinogenesis. CONCLUSION: Our results showed that many miRNAs changed their expression to respond the exposure of the genotoxic carcinogen ENU and the number and magnitude of the changes were highest at PTDs 7 to 15. Thus, one to two weeks after the exposure is the best time for miRNA expression sampling.

Gene expression changes associated with xenobiotic metabolism pathways in mice exposed to acrylamide.

The discovery of acrylamide (AA) in a variety of fried foods has raised public health concerns. In this study, groups of male mice were administered 500 mg/L AA in drinking water for 3 weeks, and gene expression changes were evaluated in the livers of AA-treated mice within 24 hr of the last treatment. When a two-fold cutoff value and a P-value less than 0.05 were selected, 696 genes (233 up-regulated and 463 down-regulated) were identified as differentially expressed genes in AA-treated mice when compared with the controls. Gene ontology analysis revealed that the principle pathways affected by AA were xenobiotic metabolism by cytochrome P450 (CYPs) and glutathione metabolism, suggesting that drug and/or xenobiotic metabolism is most affected by exposure. The results provide more information about AA metabolism and further insight into the molecular mechanisms involved in AA-induced toxicity.

Cross-platform comparison of SYBR Green real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC) study.

BACKGROUND: The MicroArray Quality Control (MAQC) project evaluated the inter- and intra-platform reproducibility of seven microarray platforms and three quantitative gene expression assays in profiling the expression of two commercially available Reference RNA samples (Nat Biotechnol 24:1115-22, 2006). The tested microarrays were the platforms from Affymetrix, Agilent Technologies, Applied Biosystems, GE Healthcare, Illumina, Eppendorf and the National Cancer Institute, and quantitative gene expression assays included TaqMan Gene Expression PCR Assay, Standardized (Sta) RT-PCRtrade mark and QuantiGene. The data showed great consistency in gene expression measurements across different microarray platforms, different technologies and test sites. However, SYBR Green real-time PCR, another common technique utilized by half of all real-time PCR users for gene expression measurement, was not addressed in the MAQC study. In the present study, we compared the performance of SYBR Green PCR with TaqMan PCR, microarrays and other quantitative technologies using the same two Reference RNA samples as the MAQC project. We assessed SYBR Green real-time PCR using commercially available RT2 Profilertrade mark PCR Arrays from SuperArray, containing primer pairs that have been experimentally validated to ensure gene-specificity and high amplification efficiency. RESULTS: The SYBR Green PCR Arrays exhibit good reproducibility among different users, PCR instruments and test sites. In addition, the SYBR Green PCR Arrays have the highest concordance with TaqMan PCR, and a high level of concordance with other quantitative methods and microarrays that were evaluated in this study in terms of fold-change correlation and overlap of lists of differentially expressed genes. CONCLUSION: These data demonstrate that SYBR Green real-time PCR delivers highly comparable results in gene expression measurement with TaqMan PCR and other high-density microarrays.

Analysis of gene expression changes in relation to toxicity and tumorigenesis in the livers of Big Blue transgenic rats fed comfrey (Symphytum officinale).

BACKGROUND: Comfrey is consumed by humans as a vegetable and a tea, and has been used as an herbal medicine for more than 2000 years. Comfrey, however, is hepatotoxic in livestock and humans and carcinogenic in experimental animals. Our previous study suggested that comfrey induces liver tumors by a genotoxic mechanism and that the pyrrolizidine alkaloids in the plant are responsible for mutation induction and tumor initiation in rat liver. RESULTS: In this study, we identified comfrey-induced gene expression profile in the livers of rats. Groups of 6 male transgenic Big Blue rats were fed a basal diet and a diet containing 8% comfrey roots, a dose that resulted in liver tumors in a previous carcinogenicity bioassay. The animals were treated for 12 weeks and sacrificed one day after the final treatment. We used a rat microarray containing 26,857 genes to perform genome-wide gene expression studies. Dietary comfrey resulted in marked changes in liver gene expression, as well as in significant decreases in the body weight and increases in liver mutant frequency. When a two-fold cutoff value and a P-value less than 0.01 were selected, 2,726 genes were identified as differentially expressed in comfrey-fed rats compared to control animals. Among these genes, there were 1,617 genes associated by Ingenuity Pathway Analysis with particular functions, and the differentially expressed genes in comfrey-fed rat livers were involved in metabolism, injury of endothelial cells, and liver injury and abnormalities, including liver fibrosis and cancer development. CONCLUSION: The gene expression profile provides us a better understanding of underlying mechanisms for comfrey-induced hepatic toxicity. Integration of gene expression changes with known pathological changes can be used to formulate a mechanistic scheme for comfrey-induced liver toxicity and tumorigenesis.

Comparison of basal gene expression profiles and effects of hepatocarcinogens on gene expression in cultured primary human hepatocytes and HepG2 cells.

Toxicogenomics is a relatively new discipline of toxicology. Microarrays and bioinformatics tools are being used successfully to understand the effects of toxicants on in vivo and in vitro model systems, and to gain a better understanding of the relevance of in vitro models commonly used in toxicological studies. In this study, cDNA filter arrays were used to determine the basal expression patterns of human cultured primary hepatocytes from different male donors; compare the gene expression profile of HepG2 to that of primary hepatocytes; and analyze the effects of three genotoxic hepatocarcinogens; aflatoxin B(1) (AFB(1)), 2-acetylaminofluorene (2AAF), and dimethylnitrosamine (DMN), as well as one non-gentoxic hepatotoxin, acetaminophen (APAP) on gene expression in both in vitro systems. Real-time PCR was used to verify differential gene expression for selected genes. Of the approximately 31,000 genes screened, 3-6% were expressed in primary hepatocytes cultured on matrigel for 16 h. Of these genes, 867 were expressed in cultured hepatocytes from all donors. HepG2 cells expressed about 98% of the genes detectable in cultured primary hepatocytes, however, 31% of the HepG2 transcriptome was unique to the cell line. A number of these genes are expressed in human liver but expression is apparently lost during culture. There was considerable variability in the response to chemical carcinogen exposure in primary hepatocytes from different donors. The transcription factors, E2F1 and ID1 mRNA were increased three-fold and six-fold (P < 0.05, P < 0.01), respectively, in AFB(1) treated primary human hepatocytes but were not altered in HepG2. ID1 expression was also increased by dimethylnitrosamine, acetylaminofluorene and acetaminophen in both primary hepatocytes and HepG2. Identification of genes that are expressed in primary hepatocytes from most donors, as well as those genes with variable expression, will aid in understanding the variability in human reactions to drugs and chemicals. This study suggests that identification of biomarkers of exposure to some chemicals may be possible in the human through microarray analysis, despite the variability in responses.

Phytoestrogens and mycoestrogens bind to the rat uterine estrogen receptor.

Consumption of phytoestrogens and mycoestrogens in food products or as dietary supplements is of interest because of both the potential beneficial and adverse effects of these compounds in estrogen-responsive target tissues. Although the hazards of exposure to potent estrogens such as diethylstilbestrol in developing male and female reproductive tracts are well characterized, less is known about the effects of weaker estrogens including phytoestrogens. With some exceptions, ligand binding to the estrogen receptor (ER) predicts uterotrophic activity. Using a well-established and rigorously validated ER-ligand binding assay, we assessed the relative binding affinity (RBA) for 46 chemicals from several chemical structure classes of potential phytoestrogens and mycoestrogens. Although none of the test compounds bound to ER with the affinity of the standard, 17beta-estradiol (E(2)), ER binding was found among all classes of chemical structures (flavones, isoflavones, flavanones, coumarins, chalcones and mycoestrogens). Estrogen receptor relative binding affinities were distributed across a wide range (from approximately 43 to 0.00008; E(2) = 100). These data can be utilized before animal testing to rank order estimates of the potential for in vivo estrogenic activity of a wide range of untested plant chemicals (as well as other chemicals) based on ER binding.