Investigating the Different Mechanisms of Genotoxic and Non-Genotoxic Carcinogens by a Gene Set Analysis

Based on the process of carcinogenesis, carcinogens are classified as either genotoxic or non-genotoxic. In contrast to non-genotoxic carcinogens, many genotoxic carcinogens have been reported to cause tumor in carcinogenic bioassays in animals. Thus evaluating the genotoxicity potential of chemicals is important to discriminate genotoxic from non-genotoxic carcinogens for health care and pharmaceutical industry safety. Additionally, investigating the difference between the mechanisms of genotoxic and non-genotoxic carcinogens could provide the foundation for a mechanism-based classification for unknown compounds. In this study, we investigated the gene expression of HepG2 cells treated with genotoxic or non-genotoxic carcinogens and compared their mechanisms of action. To enhance our understanding of the differences in the mechanisms of genotoxic and non-genotoxic carcinogens, we implemented a gene set analysis using 12 compounds for the training set (12, 24, 48 h) and validated significant gene sets using 22 compounds for the test set (24, 48 h). For a direct biological translation, we conducted a gene set analysis using Globaltest and selected significant gene sets. To validate the results, training and test compounds were predicted by the significant gene sets using a prediction analysis for microarrays (PAM). Finally, we obtained 6 gene sets, including sets enriched for genes involved in the adherens junction, bladder cancer, p53 signaling pathway, pathways in cancer, peroxisome and RNA degradation. Among the 6 gene sets, the bladder cancer and p53 signaling pathway sets were significant at 12, 24 and 48 h. We also found that the DDB2, RRM2B and GADD45A, genes related to the repair and damage prevention of DNA, were consistently up-regulated for genotoxic carcinogens. Our results suggest that a gene set analysis could provide a robust tool in the investigation of the different mechanisms of genotoxic and non-genotoxic carcinogens and construct a more detailed understanding of the perturbation of significant pathways.     

Development and validation of a high-content screening in vitro micronucleus assay in CHO-k1 and HepG2 cells

In the present study an automated image analysis assisted in vitro micronucleus assay was developed with the rodent cell line CHO-k1 and the human hepatoma cell line HepG2, which are both commonly used in regulatory genotoxicity assays. The HepG2 cell line was chosen because of the presence in these cells of a functionally active p53 protein, a functionally competent DNA-repair system, active enzymes for phase-I and -II metabolism, and an active Nrf2 electrophile responsive system. These properties may result in an assay with a high predictivity for in vivo genotoxicity. The assays with CHO-k1 and HepG2 cells were both evaluated by testing a set of compounds recommended by the European Centre for the Validation of Alternative Methods (ECVAM), among which are in vivo genotoxins and non-genotoxins. The CHO-k1 cell line showed a high sensitivity (percentage of genotoxic compounds that gave a positive result: 80%; 16/20) and specificity (percentage of non-genotoxic compounds that came out negative: 88%; 37/42). Although the sensitivity of the HepG2 cell line was lower (60%; 12/20), the specificity was high (88%; 37/42). These results were confirmed by testing an additional series of 16 genotoxic compounds. For both the CHO-k1 and the HepG2 cell line it was possible to size-classify micronuclei, enabling distinguishing aneugens from clastogens. It is concluded that two high-throughput micronucleus assays were developed that can detect genotoxic potential and allow differentiation between clastogens and aneugens. The performance scores of the CHO-k1 and HepG2 cell lines for in vivo genotoxicity were high. Application of these assays in the early discovery phase of drug development may prove to be a useful strategy to assess genotoxic potential at an early stage.     

Genotoxicity is modulated by ascorbic acid: Studies using the wing spot test in Drosophila

The ability of ascorbic acid (Vitamin C) to modulate the genotoxic action of several mutagens was investigated in the wing spot test of Drosophila melanogaster. In this assay, 3-day-old transheterozygous larvae for the multiple wing hairs (mwh, 3-0.3) and flare (flr, 3-38.8) genes were treated with three reference mutagenic compounds, namely cobalt chloride (CoCl₂), 4-nitroquinoline 1-oxide (4-NQO) and potassium dichromate (K₂Cr₂O₇). The results obtained show that the three reference mutagens tested were clearly genotoxic in the Drosophila wing somatic mutation and recombination test (SMART). None of the three concentrations tested of ascorbic acid (25, 75 and 250 mM) induced significant increases in the frequency of the mutant clones recorded. When co-treatment experiments with ascorbic acid were carried out, different results were found. Thus, ascorbic acid was effective in reducing the genotoxicity of K₂Cr₂O₇ virtually to the control level; on the contrary, it did not show any antigenotoxic effect on the genotoxicity of 4-NQO. Finally, co-treatments with CoCl₂ and ascorbic acid show a significant increase in the frequency of mutant clones over the values obtained with CoCl₂ alone.     

Combined Genotoxic Effects of a Polycyclic Aromatic Hydrocarbon (B(a)P) and an Heterocyclic Amine (PhIP) in Relation to Colorectal Carcinogenesis

Colorectal neoplasia is the third most common cancer worldwide. Environmental factors such as diet are known to be involved in the etiology of this cancer. Several epidemiological studies have suggested that specific neo-formed mutagenic compounds related to meat consumption are an underlying factor involved in the association between diet and colorectal cancer. Heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs) are known mutagens and possible human carcinogens formed at the same time in meat during cooking processes. We studied the genotoxicity of the model PAH benzo(a)pyrene (B(a)P) and HCA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), alone or in mixture, using the mouse intestinal cell line Apc^Min/+, mimicking the early step of colorectal carcinogenesis, and control Apc^+/+ cells. The genotoxicity of B(a)P and PhIP was investigated using both cell lines, through the quantification of B(a)P and PhIP derived DNA adducts, as well as the use of a genotoxic assay based on histone H2AX phosphorylation quantification. Our results demonstrate that heterozygous Apc mutated cells are more effective to metabolize B(a)P. We also established in different experiments that PhIP and B(a)P were more genotoxic on Apc^Min/+ cells compared to Apc^+/+. Moreover when tested in mixture, we observed a combined genotoxicity of B(a)P and PhIP on the two cell lines, with an increase of PhIP derived DNA adducts in the presence of B(a)P. Because of their genotoxic effects observed on heterozygous Apc mutated cells and their possible combined genotoxic effects, both B(a)P and PhIP, taken together, could be implicated in the observed association between meat consumption and colorectal cancer.     

Antimutagenicity of hops (Humulus lupulus L.): bioassay-directed fractionation and isolation of xanthohumol

Bioassay-directed fractionation with a Salmonella/microsomal assay against the food borne mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was used to identify antimutagenic components of hops. Hops pellets extracted with diethylether showed antimutagenic activity against mutations induced by IQ. Fractionation of the diethylether extract (DE) by column chromatography, followed by semi-preparative HPLC yielded two fractions (E4b and E4d) with strong antimutagenic activity against IQ induced mutations. Separation of fraction E4b resulted in inactive fractions, while fraction E4d has been identified to be xanthohumol. In mammalian test system with human hepatoma HepG2 cells fraction E4d at 10 μg/ml completely prevented formation of IQ induced DNA damage. These results indicate that xanthohumol is a very promising potential protective agent against genotoxicity of food borne carcinogens, which warrants further investigation.     

In vivo Comet assay on isolated kidney cells to distinguish genotoxic carcinogens from epigenetic carcinogens or cytotoxic compounds

The objective of this study was to determine the ability of the alkaline in vivo Comet assay (pH > 13) to distinguish genotoxic carcinogens from epigenetic carcinogens when performed on freshly isolated kidney cells and to determine the possible interference of cytotoxicity by assessing DNA damage induced by renal genotoxic, epigenetic or toxic compounds after enzymatic isolation of kidney cells from OFA Sprague–Dawley male rats. The ability of the Comet assay to distinguish (1) genotoxicity versus cytotoxicity and (2) genotoxic versus non-genotoxic (epigenetic) carcinogens, was thus investigated by studying five known genotoxic renal carcinogens acting through diverse mechanisms of action, i.e. streptozotocin, aristolochic acids, 2-nitroanisole, potassium bromate and cisplatin, two rodent renal epigenetic carcinogens: d-limonene and ciclosporine and two nephrotoxic compounds: streptomycin and indomethacin. Animals were treated once with the test compound by the appropriate route of administration and genotoxic effects were measured at the two sampling times of 3–6 and 22–26 h after treatment. Regarding the tissue processing, the limited background level of DNA migration observed in the negative control groups throughout all experiments demonstrated that the enzymatic isolation method implemented in the current study is appropriate. On the other hand, streptozotocin, 20 mg/kg, used as positive reference control concurrently to each assay, caused a clear increase in the mean Olive Tail Moment median value, which allows validating the current methodology.Under these experimental conditions, the in vivo rodent Comet assay demonstrated good sensitivity and good specificity: all the five renal genotoxic carcinogens were clearly detected in at least one expression period either directly or indirectly, as in the case of cisplatin: for this cross-linking agent, the significant decrease in DNA migration observed under standard electrophoresis conditions was clearly amplified when the duration of electrophoresis was increased up to 40 min. In contrast, epigenetic and nephrotoxic compounds failed to induce any signifcant increase in DNA migration. In conclusion, the in vivo rodent Comet assay performed on isolated kidney cells could be used as a tool to investigate the genotoxic potential of a test compound if neoplasic/preneoplasic changes occur after subchronic or chronic treatments, in order to determine the role of genotoxicity in tumor induction. Moreover, the epigenetic carcinogens and cytotoxic compounds displayed clearly negative responses in this study. These results allow excluding a DNA direct-acting mechanism of action and can thus suggest that a threshold exists. Therefore, the current in vivo rodent Comet assay could contribute to elucidate an epigenetic mechanism and thus, to undertake a risk assessment associated with human use, depending on the exposure level.     

Prevalence of genotoxic chemicals among animal and human carcinogens evaluated in the IARC Monograph series.

To determine whether genotoxic and non-genotoxic carcinogens contribute similarly to the cancer burden in humans, an analysis was performed on agents that were evaluated in Supplements 6 and 7 to the IARC Monographs for their carcinogenic effects in humans and animals and for the activity in short-term genotoxicity tests. The prevalence of genotoxic carcinogens on four groups of agents, consisting of established human carcinogens (group 1, n = 30), probable human carcinogens (group 2A, n = 37), possible human carcinogens (group 2B, n = 113) and on agents with limited evidence of carcinogenicity in animals (a subset of group 3, n = 149) was determined. A high prevalence in the order of 80 to 90% of genotoxic carcinogens was found in each of the groups 1, 2A and 2B, which were also shown to be multi-species/multi-tissues carcinogens. The distribution of carcinogenic potency in rodents did not reveal any specific characteristic of the human carcinogens in group 1 that would differentiate them from agents in groups 2A, 2B and 3. The results of this analysis indicate that (a) an agent with unknown carcinogenic potential showing sufficient evidence of activity in in vitro/in vivo genotoxicity assays (involving as endpoints DNA damage and chromosomal/mutational damage) may represent a hazard to humans; and b) an agent showing lack of activity in this spectrum of genotoxicity assays should undergo evaluation for carcinogenicity by rodent bioassay, in view of the present lack of validated short-term tests for non-genotoxic carcinogens. Overall, this analysis implies that genotoxic carcinogens add more to the cancer burden in man than non-genotoxic carcinogens. Thus, identification of such genotoxic carcinogens and subsequent lowering of exposure will remain the main goal for primary cancer prevention in man.     

The overexpression of GRASP might inhibit cell proliferation and invasion in hepatocellular carcinoma

This study aimed to validate the methylation of key genes in hepatocellular carcinoma (HCC) screened by bioinformatics analysis and explore whether they affected HCC cell proliferation, migration, and invasion. Using The Cancer Genome Atlas (TCGA) database, HCC-related differentially methylated positions (DMPs) were screened, genes corresponding to DMPs were selected, and prognosis-related genes were identified. A representative DMP was used to divide the DMPs into hyper- and hypomethylated groups. Expression of key genes in cell lines was detected using quantitative real-time polymerase chain reaction and western blot analysis. After treatment of HepG2 cells with 5-Aza-2′-deoxycytidine (5-Aza-DC), gene expression was observed. Bisulfite sequencing PCR assay was used to detect methylation frequency. Overexpressed GRASP lentiviral vectors were constructed to analyze their influence on cell proliferation, migration, and invasion using cell counting kit-8 and transwell assays. Forty-three HCC prognosis-related genes were screened using the TCGA database. cg00249511 (SCT) was used to divide the DMPs into hyper- and hypomethylated groups, distinguishing between high- and low-risk samples. The prognosis survival model constructed using 12 genes revealed the prognosis type. GRASP messenger RNA was downregulated in HepG2 and upregulated after 5-Aza-DC treatment. In HCC tissues, methylation frequency of GRASP was upregulated. GRASP overexpression inhibited HepG2 cell proliferation, invasion, and G-CSFR expression. Thus, GRASP might be a prognosis-related gene controlled by methylation.     

The Escherichia coli WP2/WP100 rec assay for detection of potential chemical carcinogens

46 chemicals of various classes and structures, including 30 known animal carcinogens, were evaluated for genotoxic effects using the Escherichia coli rec assay with strains WP2 (wild-type) and WP100 (uvrA⁻¹recA⁻) in qualitative and quantitative spot tests and in quantitative suspension tests. The rec assay detected 17 of 30 known carcinogens as genotoxic agents, including mitomycin C and diethylnitrosamine, both negative in the Salmonella/Ames test as utilized in these studies. The rec assay in conjunction with the Salmonella/Ames test 30 known carcinogens as genotoxic agents. Azo/aminoazo carcinogens showed little genotoxicity, and the aromatic amine 2-acetylaminofluorene was non-genotoxic in the rec assay. The rec assay was more effective than pol tests with E. coli strains W3110/p3478 and strains WP2/WP67. Effectiveness of the rec assay was related to the DNA repair-defective nature of the uvrA⁻ recA⁻ genotype of strain WP100.     

Alleviation of free radical mediated oxidative and genotoxic effects of cadmium by farnesol in Swiss albino mice

Abstract

Farnesol is an isoprenoid found in essential oils of ambrette seeds, citronella and in various aromatic plants. Exposure to cadmium from various sources affects the renal system adversely and Cd is an established genotoxic agent. In the present study, we evaluated the antigenotoxic and antioxidant efficacy of farnesol against cadmium chloride (CdCl₂)-induced renal oxidative stress and genotoxicity in Swiss albino mice. Single, intraperitoneal doses of CdCl₂(5 mg/kg body weight) for 24 h resulted in a significant (P < 0.001) increase in chromosomal aberration and micronuclei formation. The oral administration of farnesol at two doses (1% and 2% per kg body weight) for seven consecutive days showed significant (P < 0.05) suppression of the genotoxic effects of CdCl₂ in the modulator groups. To study the mechanism by which farnesol exerts its antigenotoxic potential, enzymes involved in metabolism and detoxification were estimated. CdCl₂ intoxication adversely affected the renal antioxidant armory and increased TBARS formation and xanthine oxidase levels significantly (P < 0.001). Farnesol showed a significant (P < 0.001) recovery in antioxidant status viz, GSH content (and its dependent enzymes) and catalase activity. Farnesol pretreatment in CdCl₂-intoxicated mice showed marked (P < 0.001) suppression of TBARS' formation and XO activity. Our results support the conclusion that the anticlastogenic effect of farnesol could be due to restoration of antioxidants and inhibition of oxidative damage.     

DNA lesions and cytogenetic changes induced by N-nitrosomorpholine in HepG2, V79 and VH10 cells: the protective effects of Vitamins A, C and E

Introduction: N-Nitrosomorpholine (NMOR), present in the workplace of tyre chemical factories, is a known hepatocarcinogen. This compound belongs to the group of N-nitrosamines, which are indirect-acting and require metabolic activation. However, the mechanism of its carcinogenic effect is not completely clear. Aims: The objective of this study was (i) to compare the DNA-damaging and clastogenic effects of NMOR in three cell lines (HepG2, V79 and VH10) with different levels of metabolizing enzymes and (ii) to determine the protective effects of Vitamins A, C and E against deleterious effects of NMOR. Methods: The exponentially growing cells were pre-treated with Vitamins A, C and E and treated with NMOR. Genotoxic effects of NMOR were evaluated by single-cell gel electrophoresis (SCGE, comet assay), while the chromosomal aberration assay was used for the study of clastogenic effects. Key results: NMOR-induced a significant dose-dependent increase of DNA damage as analyzed by SCGE, but the extent of DNA migration in the electric field was unequal in the different cell lines. Although the results obtained by SCGE confirmed the genotoxicity of NMOR in all cell lines studied, the number of chromosomal aberrations was significantly increased only in HepG2 and V79 cells, while no changes were observed in VH10 cells. In HepG2 cells pre-treated with Vitamins A, C and E we found a significant decrease of the percentage of tail DNA induced by NMOR. The reduction of the clastogenic effects of NMOR was observed only after pretreatment with Vitamins A and E; Vitamin C did not alter the frequency of NMOR-induced chromosomal aberrations under the experimental conditions of this study. Conclusions: The fat-soluble Vitamins A and E, which are dietary constituents, reduce the harmful effects of N-nitrosomorpholine in human hepatoma cells HepG2, which are endowed with the maximal capacity for metabolic activation of several drugs.     

Genotoxicity of municipal landfill leachate on root tips of Vicia faba

The genotoxicity of municipal landfill leachate was studied using the Vicia faba root-tip cytogenetic bioassay. Results show that landfill leachates collected in different seasons decreased the mitotic index (MI) and caused significant increases of micronucleus (MN) frequencies and anaphase aberration (AA) frequencies in a concentration-dependent manner (concentration expressed as ‘chemical oxygen demand’ measured by the method of potassium dichromate oxidation (COD_Cr)). In addition, a seasonal difference in genotoxicity induced by leachate was observed. The results confirm that leachate is a genotoxic agent in plant cells and imply that exposure to leachate in the aquatic environment may pose a potential genotoxic risk to organisms. The results also show that the V. faba cytogenetic bioassay is efficient, simple and reproducible in genotoxicity studies of leachate, and that there is a correlation between the genotoxicity and the chemical measurement (COD_Cr) of leachate.     

Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis

Background

The multi-step process of carcinogenesis can be more fully understood by characterizing gene expression changes induced in cells by carcinogens. In this study, expression microarrays were used to monitor the activity of 18,224 cDNA clones in MCF-7 and HepG2 cells exposed to the carcinogen benzo(a)pyrene (BaP) or its non-carcinogenic isomer benzo(e)pyrene (BeP). Time and concentration gene expression effects of BaP exposure have been assessed and linked to other measures of cellular stress to aid in the identification of novel genes/pathways involved in the cellular response to genotoxic carcinogens.

Results

BaP (0.25–5.0 μM; 6–48 h exposure) modulated 202 clones in MCF-7 cells and 127 in HepG2 cells, including 27 that were altered in both. In contrast, BeP did not induce consistent gene expression changes at the same concentrations. Significant time- and concentration-dependent responses to BaP were seen in both cell lines. Expression changes observed in both cell lines included genes involved in xenobiotic metabolism (e.g., CYP1B1, NQO1, MGST1, AKR1C1, AKR1C3,CPM), cell cycle regulation (e.g., CDKN1A), apoptosis/anti-apoptosis (e.g., BAX, IER3), chromatin assembly (e.g., histone genes), and oxidative stress response (e.g., TXNRD1). RTqPCR was used to validate microarray data. Phenotypic anchoring of the expression data to DNA adduct levels detected by ³²P-postlabelling, cell cycle data and p53 protein expression identified a number of genes that are linked to these biological outcomes, thereby strengthening the identification of target genes. The overall response to BaP consisted of up-regulation of tumour suppressor genes and down-regulation of oncogenes promoting cell cycle arrest and apoptosis. Anti-apoptotic signalling that may increase cell survival and promote tumourigenesis was also evident.

Conclusion

This study has further characterised the gene expression response of human cells after genotoxic insult, induced after exposure to concentrations of BaP that result in minimal cytotoxiCity. We have demonstrated that investigating the time and concentration effect of a carcinogen on gene expression related to other biological end-points gives greater insight into cellular responses to such compounds and strengthens the identification of target genes.     

Genotoxic effects of a complex mixture adsorbed onto ambient air particles on human cells in vitro; the effects of Vitamins E and C

Genotoxicity of complex mixtures of organic compounds adsorbed onto ambient air particles (extractable organic matter, EOM) collected in Teplice (Czech Republic) as well as genotoxicity of the indirectly acting carcinogens benzo[a]pyrene (B[a]P) and 5,9-dimethyl-7H-dibenzo[c,g]carbazole (5,9-diMeDBC) was studied in human HepG2 and Caco-2 cells cultured in vitro. The level of DNA breaks was detected by conventional single-cell gel electrophoresis (alkaline comet assay). The level of DNA breaks+oxidative DNA lesions was assessed by modified single-cell gel electrophoresis. The indirectly acting chemical carcinogens studied were able to induce DNA breaks as well as oxidative DNA damage in both cell lines, but stronger DNA-damaging effects were observed in HepG2 cells, which contain a higher level of metabolic enzymes. Treatment of cells with the complex mixtures showed a dose-dependent increase of DNA breaks in HepG2 cells as well as in Caco-2 cells, with seasonal differences. Winter samples of EOM from Teplice (TP-W) were more effective in inducing DNA damage than summer samples (TP-S). Both mixtures caused significant oxidative DNA damage in HepG2 cells. The effect was less evident in cells treated with higher concentrations of TP-W, since the comet assay is limited by saturation at a higher level of DNA damage. Possible reduction of B[a]P-, 5,9-diMeDBC- or EOM-induced DNA damage by Vitamins E and C was evaluated in HepG2 cells only. Pre-treatment of these cells with either one of the vitamins considerably reduced the levels of both DNA breaks and oxidative DNA lesions induced by all compounds investigated.     

Possible involvement of oxidative stress in potassium bromate-induced genotoxicity in human HepG2 cells

Potassium bromate (KBrO₃, PB) is a by-product of ozone used as disinfectant in drinking water. And PB is also a widely used food additive. However, there is little known about its adverse effects, in particular those related to its genotoxicity in humans. The aim of this study was to investigate the genotoxic effects of PB and the underlying mechanisms, using human hepatoma cell line, HepG2. Exposure of the cells to PB caused a significant increase of DNA migration in single cell gel electrophoresis (SCGE) assay and micronuclei (MN) frequencies in micronucleus test (MNT) at all tested concentrations (1.56–12.5 mM and 0.12–1 mM), which suggested that PB-mediated DNA strand breaks and chromosome damage. To indicate the role of antioxidant in those effects, DNA migration was monitored by pre-treatment with hydroxytyrosol (HT) as an antioxidant in SCGE assay. It was found that DNA migration with pre-treatment of HT was dramatically decreased. To elucidate the genotoxicity mechanisms, the study monitored the levels of reactive oxygen species (ROS), glutathione (GSH) and 8-hydroxydeoxyguanosine (8-OHdG). PB was shown to induce ROS production (12.5 mM), GSH depletion (1.56–12.5 mM) and 8-OHdG formation (6.25–12.5 mM) in HepG2 cells. Moreover, lysosomal membrane stability and mitochondrial membrane potential were further studied for the mechanisms of PB-induced genotoxicity. A significant increase was found in the range of 6.25–12.5 mM in lysosomal membrane stability assay. However, under these PB concentrations, we were not able to detect the changes of mitochondrial membrane potential. These results suggest that PB exerts oxidative stress and genotoxic effects in HepG2 cells, possibly through the mechanisms of lysosomal damage, an earlier event preceding the oxidative DNA damage.     

The activities of several detoxication enzymes are differentially induced by juices of garden cress, water cress and mustard in human HepG2 cells

It has been previously demonstrated in a human-derived hepatoma cell line (HepG2) that juices from cruciferous vegetables protect against the genotoxicity caused by dietary carcinogens. HepG2 cells possess different enzymes involved in the biotransformation of xenobiotics. Therefore, we investigated the effect of cruciferous juices on the activities of CYP 1A and several phase II enzymes in this cell model. For each experiment, 1 × 10⁶ cells were seeded on Petri dishes. After 2 days, the juices (0.5–8 μl/ml of culture medium) were added for 48 h prior to cell harvesting. The addition of juice from water cress (Nasturtium officinalis R. Br) significantly increased the activities of ethoxyresorufin-O-deethylase at high doses only and NAD(P)H-quinone reductase in a dose-dependent manner (1.8- and 5-fold, respectively). The addition of juice from garden cress (Lepidum sativum L.) significantly increased the activities of NAD(P)H-quinone reductase and UDP-glucuronosyl-transferase with a maximal effect around the dose of 2 μl/ml juice (1.4- and 1.2-fold, respectively) while the other enzymes were not altered. Mustard (Sinapis alba L.) juice increased the activities of NAD(P)H-quinone reductase (2.6-fold at the dose of 8 μl/ml), and N-acetyl-transferase (1.4-fold at the dose of 8 μl/ml) in a dose-dependent manner while a maximal induction of UDP-glucuronosyl-transferase was obtained with a dose of 2 μl/ml (1.8-fold). These observations show that the three juices have different induction profiles: only water cress acted as a bifunctional inducer by enhancing both phase I and phase II enzymes. As a consequence, each juice may preferentially inhibit the genotoxicity of specific compounds.     

The P2Y2 Nucleotide Receptor Mediates the Proliferation and Migration of Human Hepatocellular Carcinoma Cells Induced by ATP

ATP is an abundant biochemical component of the tumor microenvironment and a physiologic ligand for the P2Y₂ nucleotide receptor (P2Y₂R). In this study, we investigated the effect of ATP on the cellular behavior of human hepatocellular carcinoma (HCC) cells and the role of P2Y₂R in ATP action and aimed to find a new therapeutic target against HCC. The experiments were performed in native isolated human HCC cells, normal hepatocytes, human HCC cell lines, and nude mice. We found that the mRNA and protein expression levels of P2Y₂R in native human HCC cells and the human HCC cell lines HepG2 and BEL-7404 were enhanced markedly compared with human normal hepatocytes and the normal hepatocyte line LO2, respectively. ATP induced intracellular Ca²⁺ increases in HCC cells and promoted the proliferation and migration of HCC cells and the growth of HCC in nude mice. The P2Y receptor antagonist suramin, P2Y₂R-specific shRNA, the store-operated calcium channel inhibitors 2-aminoethoxydiphenyl borate (2-APB) and 1-(β-3-(4-methoxy-phenyl) propoxyl-4-methoxyphenethyl)1H-imidazole-hydrochloride ({"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365), and stromal interaction molecule (STIM1)-specific shRNA inhibited the action of ATP on HCC cells. In conclusion, P2Y₂R mediated the action of ATP on the cellular behavior of HCC cells through store-operated calcium channel-mediated Ca²⁺ signaling, and targeting P2Y₂R may be a promising therapeutic strategy against human HCC.     

Protection by beverages, fruits, vegetables, herbs, and flavonoids against genotoxicity of 2-acetylaminofluorene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in metabolically competent V79 cells

Chinese hamster lung fibroblasts, genetically engineered for the expression of rat cytochrome P450 dependent monooxygenase 1A2 and rat sulfotransferase 1C1 (V79-rCYP1A2-rSULT1C1 cells), were utilized to check for possible protective effects of beverages of plant origin, fruits, vegetables, and spices against genotoxicity induced by 2-acetylaminofluorene (AAF) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Antigenotoxic activities of juices from spinach and red beets against AAF could be monitored with similar effectivity by the HPRT-mutagenicity test (IC₅₀=0.64%; 2.57%) and alkaline single cell gel electrophoresis (comet assay; IC₅₀=0.12%; 0.89%) which detects DNA strand breaks and abasic sites. Applying the comet assay, genotoxicity of PhIP could, however, be demonstrated only in the presence of hydroxyurea and 1-[β- -arabinofuranosyl]cytosine, known inhibitors of DNA repair synthesis. As expected, AAF and PhIP were unable to induce any genotoxic effects in the parent V79 cells. Genotoxic activity of PhIP was strongly reduced in a dose-related manner by green tea and red wine, by blueberries, blackberries, red grapes, kiwi, watermelon, parsley, and spinach, while two brands of beer, coffee, black tea, rooibos tea, morellos, black-currants, plums, red beets, broccoli (raw and cooked), and chives were somewhat less active. One brand of beer was only moderately active while white wine, bananas, white grapes, and strawberries were inactive. Similarly, genotoxicity of AAF was strongly reduced by green, black, and rooibos tea, red wine, morellos, black-currants, kiwi, watermelon, and spinach while plums, red beets, and broccoli (raw) were less potent. Broccoli cooked exerted only moderate and white wine weak antigenotoxic activity. With respect to the possible mechanism(s) of inhibition of genotoxicity, benzo[a]pyrene-7,8-dihydrodiol (BaP-7,8-OH) and N-OH-PhIP were applied as substrates for the CYP1A family and for rSULT 1C1, respectively. Morellos, black-currants, and black tea strongly reduced the genotoxicity of BaP-7,8-OH, onions, rooibos tea, and red wine were less potent while red beets and spinach were inactive. On the other hand, red beets and spinach strongly inhibited the genotoxicity of N-OH-PhIP, rooibos tea was weakly active while all other items were inactive. These results are suggestive for enzyme inhibition as mechanism of protection by complex mixtures of plant origin. Taken together, our results demonstrate that protection by beverages, fruits, and vegetables against genotoxicity of heterocyclic aromatic amines may take place within metabolically competent mammalian cells as well as under the conditions of the Salmonella/reversion assay [Food Chem. Toxicol. 32 (1994) 443; Mutat. Res. 341 (1995) 303].     

Diterpenoids from Euphorbia neriifolia and Their Related Anti‐HIV and Cytotoxic Activity

Fifteen diterpenoids ( 1 – 15 ), including three undescribed ones with ent‐atisane skeleton, eupnerias G–I ( 1 – 3 ), were obtained from Euphorbia neriifolia. Compounds 1 – 3 were established through comprehensive spectroscopic analysis. Compounds 4 and 5 exhibited obvious anti‐HIV‐1 effect, and their EC₅₀ were 6.6±3.2 and 6.4±2.5 μg mL^?1, respectively. Compound 6 exhibited moderate cytotoxicity on HepG2 and HepG2/Adr cells with IC₅₀ at 13.70 and 15.57 μm , respectively. In addition, compound 15 exhibited significant cytotoxicity on HepG2 cell lines (IC₅₀=0.01 μm ), while it did not show any cytotoxicity against HepG2/Adr cell lines.