4NQO Research Articles

Deficiency of IL-18 Aggravates Esophageal Carcinoma Through Inhibiting IFN-γ Production by CD8+T Cells and NK Cells.

To investigate the potential role of interleukin-18 (IL-18) in immunomodulation during tumorigenesis of esophageal carcinoma and elucidate the underlying molecular mechanism, we employed IL-18 knockout mice for this purpose. Carcinogen 4-nitroquinoline 1-oxide (4NQO) was administrated in drinking water to induce occurrence of esophageal squamous cell carcinoma (ESCC). T cell activation as indicated by the surface CD molecules was analyzed with flow cytometry. The serous content of interferon-γ (IFN-γ) along with other cytokines was determined by inflammatory human cytokine cytometric bead array. The cytotoxicity assay was performed by co-culture of tumor cells with immune cells and relative cell viability was determined by lactate dehydrogenase (LDH) assay. Apoptotic cells were stained with Annexin-V/propidium iodide (PI) and analyzed by flow cytometry. Cell proliferation was measured with Cell Counting Kit-8 (CCK-8) assay. Our data demonstrated that deficiency of IL-18 promoted the progression and development of 4NQO-induced ESCC. Loss of IL-18 suppressed the activation of T cells in the esophagus. Deficiency of IL-18 inhibited the IFN-γ production by CD8+ T cells and natural killer (NK) cells. Absence of IL-18 inhibited the cytotoxicity of CD8+ T cells and NK cell in vitro. Moreover, deficiency of IL-18 promoted the apoptosis of CD8+ T cells and inhibited the proliferation of CD8+ T cells in vitro. Our data elucidated the immunomodulatory role of IL-18 during tumorigenesis of ESCC, whose deficiency compromised antitumor immunity and contributed to immune escape of esophageal carcinoma. Our results also indicated the therapeutic potential of exogenous IL-18 against ESCC, which warrants further investigations.

Effects of chitosan molecular weight on its antioxidant and antimutagenic properties

Chitosan (300.0kDa) was enzymatically degraded to obtain six degraded chitosans with molecular weights (MWs) of 156.0, 72.1, 29.2, 7.1, 3.3, and 2.2kDa. The antioxidant and antimutagenic properties of all seven chitosan samples were evaluated in vitro. The antioxidant scavenging activities of chitosans on hydrogen peroxide and 2, 2-diphenyl-1-picryl hydrazyl radical, and on chelating ferrous ion were significantly increased with chitosan’s decreasing MW, with 2.2-kDa chitosan have the highest impact. All seven chitosan samples exhibited strong anitmutagenic effects against direct (4- nitroquinoline 1-oxide) and indirect (benzo[α]pyrene) mutagens in the presence of rat liver enzyme S9 mix in Salmonella typhimurium TA 98 and TA 100. The effect of MW on antimutagenicity was similar to that on antioxidant activity. The underlying mechanism for chitosan antimutagenicity proved to be its desmutagenic effect on mutagens.

Inhibition of Pro-inflammatory and Anti-apoptotic Biomarkers during Experimental Oral Cancer Chemoprevention by Dietary Black Raspberries.

Oral cancer continues to be a significant public health problem worldwide. Recently conducted clinical trials demonstrate the ability of black raspberries (BRBs) to modulate biomarkers of molecular efficacy that supports a chemopreventive strategy against oral cancer. However, it is essential that a preclinical animal model of black raspberry (BRB) chemoprevention which recapitulates human oral carcinogenesis be developed, so that we can validate biomarkers and evaluate potential mechanisms of action. We therefore established the ability of BRBs to inhibit oral lesion formation in a carcinogen-induced rat oral cancer model and examined potential mechanisms. F344 rats were administered 4-nitroquinoline 1-oxide (4NQO) (20 µg/ml) in drinking water for 14 weeks followed by regular drinking water for 6 weeks. At week 14, rats were fed a diet containing either 5 or 10% BRB, or 0.4% ellagic acid (EA), a BRB phytochemical. Dietary administration of 5 and 10% BRB reduced oral lesion incidence and multiplicity by 39.3 and 28.6%, respectively. Histopathological analyses demonstrate the ability of BRBs and, to a lesser extent EA, to inhibit the progression of oral cancer. Oral lesion inhibition by BRBs was associated with a reduction in the mRNA expression of pro-inflammatory biomarkers Cxcl1, Mif, and Nfe2l2 as well as the anti-apoptotic and cell cycle associated markers Birc5, Aurka, Ccna1, and Ccna2. Cellular proliferation (Ki-67 staining) in tongue lesions was inhibited by BRBs and EA. Our study demonstrates that, in the rat 4NQO oral cancer model, dietary administration of BRBs inhibits oral carcinogenesis via inhibition of pro-inflammatory and anti-apoptotic pathways.

Nicotine suppresses apoptosis by regulating α7nAChR/Prx1 axis in oral precancerous lesions.

Nicotine, a tumor promoter in tobacco, can increase Peroxiredoxin (Prx1) and nicotinic acetylcholine receptors (nAChRs) in oral squamous cell carcinoma (OSCC). In the present study, we investigate the effects of nicotine in oral precancerous lesions focusing on apoptosis and nAChR/Prx1 signaling. We detected expression of Prx1, α3nAChR, α7nAChR, phosphorylation of mitogen-activated protein kinases (MAPK) and apoptosis in dysplastic oral keratinocyte (DOK) cells as well as in 4-nitroquinoline 1-oxide (4NQO) or 4NQO + nicotine – induced oral precancerous lesions in Prx1 wild-type (Prx1+/+) and Prx1 knockdown (Prx1+/-) mice. In DOK cells, Prx1 knockdown and blocking α7nAChR activated apoptosis, and nicotine increased the expression of Prx1, α3nAChR and α7nAChR, and inhibited MAPK activation. Moreover, nicotine suppressed apoptosis depending on Prx1 and α7nAChR in DOK cells. In animal bioassay, nicotine and Prx1 promoted growth of 4NQO-induced precancerous lesions in mouse tongue. 4NQO plus nicotine suppressed MAPK activation in Prx1 wild-type mice but not in Prx1 knockdown mice. Our data demonstrate that nicotine inhibits cell apoptosis and promotes the growth of oral precancerous lesions via regulating α7nAChR/Prx1 during carcinogenesis of OSCC.

NQO-Induced DNA-Less Cell Formation Is Associated with Chromatin Protein Degradation and Dependent on A0A1-ATPase in Sulfolobus.

To investigate DNA damage response in the model crenarchaeon Sulfolobus islandicus, four different DNA damage agents were tested for their effects on cell death of this archaeon, including UV irradiation, methyl methanesulfonate, cisplatin, and 4-nitroquinoline 1-oxide (NQO). Cell death featured with DNA-less cell formation was revealed in DNA damage treatment with each agent. Cellular responses upon NQO treatment were characterized in details, and following sequential events were revealed, including: a modest accumulation of G1/S phase cells, membrane depolarization, proteolytic degradation of chromatin proteins, and chromosomal DNA degradation. Further insights into the process were gained from studying drugs that affect the archaeal ATP synthase, including a proton gradient uncoupler and an ATP synthase inhibitor. Whereas the proton uncoupler-mediated excess proton influx yielded cell death as observed for the NQO treatment, inhibition of ATP synthase attenuated NQO-induced membrane depolarization and DNA-less cell formation. In conclusion, the NQO-induced cell death in S. islandicus is characterized by proteolytic degradation of chromatin protein, and chromosomal DNA degradation, which probably represents a common feature for the cell death induced by different DNA damage agents.

Abstract 5264: Chemoprevention of rat oral carcinogenesis by black raspberry phytochemicals

Abstract It is expected that in 2016, there will be an estimated 48,330 new cases and 9,570 deaths due to oral cancer in the US, which is about one person every hour every day. Due to the epithelial field defects associated with oral carcinogenesis, tumor recurrence and second primary tumor incidence are common. Recently conducted Phase I Clinical trials demonstrate the ability of black raspberries (BRBs) to modulate inflammatory biomarkers of molecular efficacy that supports a chemopreventive strategy against oral cancer. From these human trials we have established a molecular signature of BRB responsive biomarkers. However, it is essential that a preclinical animal model of BRB chemoprevention which recapitulates the fundamental features of human oral carcinogenesis be developed, so that we can evaluate potential mechanisms of action and validate molecular biomarkers of BRB efficacy. We therefore established the ability of BRB to inhibit oral lesion formation in a rat model of 4-nitroquinoline 1-oxide (4NQO) induced oral carcinogenesis and examined molecular efficacy using a clinically relevant transcriptional signature. Oral cancer was induced in F344 rats using 4NQO in drinking water for 10 weeks followed by regular drinking water for 16 weeks. Rats were administered a diet containing 5 or 10 percent lyophilized BRBs. Tongue tissues were examined for lesion multiplicity, incidence and histopathological features. Clinically derived pro-inflammatory and pro-survival molecular biomarkers were also determined by real-time PCR and ELISA. Dietary administration of 5 and 10 percent BRBs reduced combined lesion incidence and multiplicity by -39.3% and -28.6% respectively. Histopathological analyses of tongue sections further demonstrate the chemopreventive ability of dietary BRB administration in the rat model. Pro-survival and pro-inflammatory biomarkers were downregulated in BRB administered 4NQO induced rats compared to untreated 4NQO induced rats. Our study shows that dietary administration of BRB inhibits oral carcinogenesis in the rat 4NQO model. The rat 4NQO model is suitable for interrogating mechanisms of action of BRB-mediated oral cancer chemoprevention. Citation Format: Steve Oghumu, Thomas Knobloch, Jennifer Ahn-Jarvis, Logan Weghorst, Kyle Horvath, Paul Geuy, Christopher Weghorst. Chemoprevention of rat oral carcinogenesis by black raspberry phytochemicals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5264. doi:10.1158/1538-7445.AM2017-5264

Abstract 1850: Development of a NOTCH1 loss of function and PIK3CA mutant transgenic model of HNSCC

Abstract Head and neck squamous cell carcinoma (HSNCC) is a debilitating disease with a poor 5-year survival rate. The use of in vivo models to study tumorigenesis and to identify potential targeted therapies, especially those that work in combination with immune checkpoint inhibitors, is essential in improving overall patient care. However, the overall number of HNSCC transgenic models with recurrent genetic lesions identified by the HNSCC TCGA project has been limited. For example, TCGA data has shown both Notch1 loss of function mutations and PIK3CA activating mutations as some of the most common in HNSCC, but bi-genic models of these lesions do not yet exist. Here, we have designed a model which utilizes K14-CreERT to drive a bi-genic model with Notch1flox and PIK3CAH1047R mutations in epithelial compartments. Characterization of this line both in the context of spontaneous and carcinogen induced tumor formation is a tool which can be utilized to better understand common mutations in HNSCC. Immunohistochemistry confirms the overexpression of PIK3CA in tamoxifen induced mice. Furthermore, early characterization of this model with chronic treatment of the smoking analogue 4-Nitroquinoline N-oxide (4NQO) in drinking water shows tumor formation on the tongue and surrounding head and neck regions in as little as 12-16 weeks. Hematoxylin and Eosin staining confirmed the squamous tumor formation, while Ki67 staining showed enhanced proliferation of the transformed cells. In the future, this model can be used to study tumor formation over time and in response to targeted inhibitors and common therapies that are being advanced as companion diagnostic strategies for either NOTCH1 deficient or PIK3CA aberrant HNSCC tumors. Further detailed characterization of this model will allow for a deeper understanding of the mechanisms which drive HNSCC and will translate to improved patient care. Citation Format: Samantha N. Devenport, Nicole L. Michmerhuizen, Elizabeth Leonard, Brittany Jewell, Jacob Swanson, Sunny Wong, Thomas E. Carey, J Chad Brenner. Development of a NOTCH1 loss of function and PIK3CA mutant transgenic model of HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1850. doi:10.1158/1538-7445.AM2017-1850

The role of NLRP3 inflammasome in 5-fluorouracil resistance of oral squamous cell carcinoma

Background5-Fluorouracil (5-FU) is a widely used drug for the therapy of cancer. However, the chemoresistance of tumor cells to 5-FU usually limits its clinical effectiveness. In this study, we explored the role of NLRP3 inflammasome in 5-FU resistance of oral squamous cell carcinoma (OSCC).MethodsThe mRNA and protein expression levels of NLRP3, Caspase1 and IL-1β in resected OSCC specimens or cell lines were measured respectively by quantitative real time-PCR (qRT-PCR) and western blot. NLRP3 and Ki-67 expression in paraffin-embedded OSCC tissues was determined by immunohistochemistry. The correlation between 5-FU treatment and the expression and activation of NLRP3 inflammasome was further examined by evaluating NLRP3 and IL-1β expression in OSCC cell lines without or with NLRP3 knocked down. Cell viabilities of OSCC cells were determined by the MTT assay. Apoptosis and intracellular reactive oxygen species (ROS) of OSCC cells induced by 5-FU were measured by the flow cytometer. The carcinogen-induced tongue squamous carcinoma mice model was established by continuous oral administration of 4-nitroquinoline 1-oxide in wild-type BALB/c, Nlrp3−/− and Caspase1−/− mice. Tumor incidence were observed and tumor area were evaluated.ResultsIn the clinical analysis, expression and activation of NLRP3 inflammasome was clearly increased in OSCC tissues of patients who received 5-FU-based chemotherapy. Multivariate Cox regression analysis revealed that this high expression was significantly correlated with tumor stage and differentiation, and was associated with poor prognosis. Moreover, 5-FU treatment increased expression and activation of NLRP3 inflammasome in OSCC cells in a cell culture system and xenograft mouse model. Silencing of NLRP3 expression significantly inhibited OSCC cell proliferation and enhanced 5-FU-induced apoptosis of OSCC cells. Further investigation showed that intracellular ROS induced by 5-FU promoted the expression and activation of NLRP3 inflammasome and increased the production of interleukin (IL)-1β, which then mediated the chemoresistance. With the carcinogen-induced OSCC model, we found less and later tumor incidence in Nlrp3−/− and Caspase1−/− mice than wild-type mice. And greater decrease of tumor area was observed in the gene deficient mice treated with 5-FU.ConclusionsOur findings suggest that NLRP3 inflammasome promoted 5-FU resistance of OSCC both in vitro and in vivo, and targeting the ROS/NLRP3 inflammasome/IL-1β signaling pathway may help 5-FU-based adjuvant chemotherapy of OSCC.

4-nitroquinoline 1-oxide誘発ラット舌癌発生過程における蛍光光学機器解析(大阪歯科大学大学院歯学研究科博士(歯学)学位論文内容要旨および論文審査結果要旨の公表)

4-nitroquinoline 1-oxide誘発ラット舌癌発生過程における蛍光光学機器解析(大阪歯科大学大学院歯学研究科博士(歯学)学位論文内容要旨および論文審査結果要旨の公表)

12 4-nitroquinoline 1-oxide誘発ラット舌癌発生過程におけるp21WAF1/Cip1の発現(第511回 大阪歯科学会例会)

12 4-nitroquinoline 1-oxide誘発ラット舌癌発生過程におけるp21WAF1/Cip1の発現(第511回 大阪歯科学会例会)

Rev7, the regulatory subunit of Polζ, undergoes UV-induced and Cul4-dependent degradation.

In eukaryotic cells, Rev7 interacts with Rev3 and functions as a regulatory subunit of Polζ, a translesion DNA synthesis (TLS) polymerase. In addition to its role in TLS, mammalian Rev7, also known as Mad2B/Mad2L2, participates in multiple cellular activities including cell cycle progression and double-strand break repair through its interaction with several proteins. Here we show that in mammalian cells, Rev7 undergoes ubiquitin/proteasome-mediated degradation upon UV irradiation in a time-dependent manner. We identified the Rev7 N-terminal destruction box as the degron and Cul4A/B as putative E3 ligases in this process. We also show that the nucleotide excision repair (NER) pathway protein HR23B physically interacts and colocalizes with Rev7 in the nuclear foci after UV irradiation and protects Rev7 from accelerated degradation. Furthermore, a similar Rev7 degradation profile was observed in cells treated with the UV-mimetic agent 4-nitroquinoline 1-oxide but not with cisplatin or camptothecin, suggesting a role of the NER pathway protein(s) in UV-induced Rev7 degradation. These data and the observation that cells deficient in Rev7 are sensitized to UV irradiation while excessive Rev7 protects cells from UV-induced DNA damage provide a new insight into the potential interplay between TLS and NER.

4 NQO誘発マウス舌癌発生過程におけるCK 19の発現について

4 NQO誘発マウス舌癌発生過程におけるCK 19の発現について

4-nitroquinoline 1-oxide誘発ラット舌癌細胞におけるp 27Kip1の分解

4-nitroquinoline 1-oxide誘発ラット舌癌細胞におけるp 27Kip1の分解

6520 4-nitroquinoline 1-oxide誘発ラット舌癌由来細胞株におけるFas抗原の発現

6520 4-nitroquinoline 1-oxide誘発ラット舌癌由来細胞株におけるFas抗原の発現

6 4-nitroquinoline 1-oxide誘発ラット舌癌におけるp53の発現について

6 4-nitroquinoline 1-oxide誘発ラット舌癌におけるp53の発現について

P53 induction and cell viability modulation by genotoxic individual chemicals and mixtures.

The binding of the p53 tumor suppression protein to DNA response elements after genotoxic stress can be quantified by cell-based reporter gene assays as a DNA damage endpoint. Currently, bioassay evaluation of environmental samples requires further knowledge on p53 induction by chemical mixtures and on cytotoxicity interference with p53 induction analysis for proper interpretation of results. We investigated the effects of genotoxic pharmaceuticals (actinomycin D, cyclophosphamide) and nitroaromatic compounds (4-nitroquinoline 1-oxide, 3-nitrobenzanthrone) on p53 induction and cell viability using a reporter gene and a colorimetric assay, respectively. Individual exposures were conducted in the absence or presence of metabolic activation system, while binary and tertiary mixtures were tested in its absence only. Cell viability reduction tended to present direct correlation with p53 induction, and induction peaks occurred mainly at chemical concentrations causing cell viability below 80%. Mixtures presented in general good agreement between predicted and measured p53 induction factors at lower concentrations, while higher chemical concentrations gave lower values than expected. Cytotoxicity evaluation supported the selection of concentration ranges for the p53 assay and the interpretation of its results. The often used 80% viability threshold as a basis to select the maximum test concentration for cell-based assays was not adequate for p53 induction assessment. Instead, concentrations causing up to 50% cell viability reduction should be evaluated in order to identify the lowest observed effect concentration and peak values following meaningful p53 induction.

Areca nut is associated with younger age of diagnosis, poor chemoradiotherapy response, and shorter overall survival in esophageal squamous cell carcinoma.

ObjectiveAreca nut chewing is carcinogenic to humans. However, little is known about the impact of areca nut chewing on esophageal squamous cell carcinoma (ESCC).MethodsWe retrospectively reviewed 286 ESCC patients who received surgery or preoperative chemoradiotherapy followed by surgery at our institution. Background characteristics including areca nut chewing history were analyzed. The 4-nitroquinoline 1-oxide (4-NQO)-induced murine ESCC model was used to test the impact of arecoline, a main constituent of areca nut, on ESCC.ResultsCompared to patients without areca nut chewing history, patients with areca nut chewing history had overall a younger age of onset (Mean age: 56.75 versus 52.68 yrs, P<0.001) and significantly worse overall survival than those without areca nut chewing history (P = 0.026). Among patients who received surgery, the overall survival rates were not significantly different between those with or without areca nut chewing history. Among patients who received preoperative chemoradiotherapy followed by surgery, those with areca nut chewing history had a significantly lower pathologic complete response rate (P = 0.002) and lower overall survival rate (P = 0.002) than those without. In the murine ESCC model, the incidence of esophageal invasive squamous cell carcinoma was 40% in mice exposed to concomitant 4-NQO and arecoline treatment for 8 weeks and 6% in mice exposed to 4-NQO only for 8 weeks (P = 0.037).ConclusionsOur results indicate that areca nut chewing history is significantly associated with younger age of onset, poor response to chemoradiotherapy, and shorter overall survival in ESCC patients. Arecoline, a main constituent of areca nut, accelerates esophageal tumorigenesis in the 4-NQO-induced murine ESCC model.

4-Nitroquinoline 1-Oxide-Induced Tongue and Esophagus Carcinogenesis in Obese and Diabetic TSOD Mice.

BackgroundObesity and diabetes mellitus are associated with lifestyle-related carcinogenesis. They are also risk factors of esophageal adenocarcinoma, but there are only a few reports on association between obesity/diabetes and development of squamous cell carcinoma in the oral cavity and esophagus. In this study, we therefore aimed to determine whether obesity and diabetes affect oral and esophageal carcinogenesis using model mice of obesity and diabetes, the Tsumura Suzuki obese diabetes (TSOD) and Tsumura Suzuki non-obesity (TSNO) control mice, which were treated with 4-nitroquinoline 1-oxide (4-NQO) to produce tongue and esophageal carcinomas.MethodsWe used 28 each of the male TSOD and TSNO mice of 8 weeks of age. They were divided into the 4-NQO-treated group (n = 20) and untreated group (n = 8). 4-NQO was administered to mice in drinking water at a dose level of 20 ppm for 8 weeks. The untreated group was given distilled water without 4-NQO. At 28 experimental weeks, histopathological examination was performed on all organs including tongue and esophagus. We performed analysis of histopathology of all organs which included buccal capsule (a tongue)/esophagus after an experiment start in 28 weeks. Fasting plasma glucose (FPG) and lipid parameters including total cholesterol (T-Cho), triglyceride (TG), high-density lipoprotein (HDL)-cholesterol and low-density lipoprotein (LDL)-cholesterol were measured and all these parameters were compared between the two genotypes. Also, mRNA expression of eight cytokines including interleukin (IL)-1β, IL-6, IL-17, interferon (IFN)-γ, keratinocyte-derived cytokine (KC), macrophage inflammatory protein (MIP)-1α, MIP-2, and tumor necrosis factor (TNF)-α in the esophageal mucosa was assayed.Results4-NQO treatment produced proliferative squamous cell lesions (dysplasia, papilloma and carcinoma) in the tongue and esophagus of both the TSOD and TSNO mice. The incidence and multiplicity of tongue tumors were 30% and 0.45 ± 0.83 in the TSOD mice and 30% and 0.40 ± 0.68 in the TSNO mice. The incidence and multiplicity of esophageal tumors were 70% and 2.25 ± 2.29 in the TSOD mice and 30% and 0.60 ± 1.14 (P < 0.01) in the TSNO mice.ConclusionOur findings indicate that the obese and diabetic TSOD mice were susceptible to 4-NQO-induced esophageal carcinogenesis, suggesting risk factors of obese and diabetes for esophageal squamous cell carcinoma. Additionally, the TSOD mice were useful as esophagus carcinogenic model. Our study first reported that 4-NQO induced esophageal cancer in mice.

The Plant Hormone Cytokinin Confers Protection against Oxidative Stress in Mammalian Cells

Modulating key dynamics of plant growth and development, the effects of the plant hormone cytokinin on animal cells gained much attention recently. Most previous studies on cytokinin effects on mammalian cells have been conducted with elevated cytokinin concentration (in the μM range). However, to examine physiologically relevant dose effects of cytokinins on animal cells, we systematically analyzed the impact of kinetin in cultured cells at low and high concentrations (1nM-10μM) and examined cytotoxic and genotoxic conditions. We furthermore measured the intrinsic antioxidant activity of kinetin in a cell-free system using the Ferric Reducing Antioxidant Power assay and in cells using the dihydroethidium staining method. Monitoring viability, we looked at kinetin effects in mammalian cells such as HL60 cells, HaCaT human keratinocyte cells, NRK rat epithelial kidney cells and human peripheral lymphocytes. Kinetin manifests no antioxidant activity in the cell free system and high doses of kinetin (500 nM and higher) reduce cell viability and mediate DNA damage in vitro. In contrast, low doses (concentrations up to 100 nM) of kinetin confer protection in cells against oxidative stress. Moreover, our results show that pretreatment of the cells with kinetin significantly reduces 4-nitroquinoline 1-oxide mediated reactive oxygen species production. Also, pretreatment with kinetin retains cellular GSH levels when they are also treated with the GSH-depleting agent patulin. Our results explicitly show that low kinetin doses reduce apoptosis and protect cells from oxidative stress mediated cell death. Future studies on the interaction between cytokinins and human cellular pathway targets will be intriguing.

The correlation between antimutagenic activity and total phenolic content of extracts of 31 plant species with high antioxidant activity.

BackgroundAntimutagenic activity of plant extracts is important in the discovery of new, effective cancer preventing agents. There is increasing evidence that cancer and other mutation-related diseases can be prevented by intake of DNA protective agents. The identification of antimutagenic agents present in plants presents an effective strategy to inhibit pathogenic processes resulting from exposure to mutagenic and/or carcinogenic substances present in the environment. There are no reports on the antimutagenic activities of the plant species investigated in this study. Many mutations related to oxidative stress and DNA damage by reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been identified in numerous human syndromes. Oxidative DNA damage plays a significant role in mutagenesis, cancer, aging and other human pathologies. Since oxidative DNA damage plays a role in the pathogenesis of several chronic degenerative diseases, the decrease of the oxidative stress could be the best possible strategy for prevention of these diseases. Antioxidant compounds can play a preventative role against mutation-related diseases, and thus have potential antimutagenic effects.MethodsThe number of antioxidant compounds present in methanol leaf extracts of 120 plant species was determined using a combination of Thin Layer Chromatography (TLC) and spraying with 2, 2-diphenyl-1-picrylhydrazyl (DPPH). The 31 most promising extracts were selected for further assays. The quantitative antioxidant activity was determined using DPPH free radical scavenging spectrophotometric assay. Total phenolic contents were determined using the Folin-Ciocalteu colorimetric assay. The mutagenicity of 31 selected extracts was determined in the Ames test using Salmonella typhimurium strains TA98 and TA100. The antimutagenicity of the plant extracts against 4-nitroquinoline 1-oxide (4-NQO) was also determined using the Ames test.ResultsOf the 120 plant extracts assayed qualitatively, 117 had some antioxidant activity. The selected 31 extracts contained well defined antioxidant compounds. These species had good DPPH free radical antioxidant activity with EC50 values ranging from 1.20 to 19.06 μg/ml. Some of the plant extracts had higher antioxidant activity than L-ascorbic acid (vitamin C). The total phenolic contents ranged from 5.17 to 18.65 mg GAE (gallic acid equivalent)/g plant extract). The total phenolic content of the plant extracts correlated well with the respective antioxidant activity of the plant extracts. No plant extract with good antioxidant activity had mutagenic activity. Several extracts had antimutagenic activity. The percentage inhibition of 4-NQO ranged from 0.8 to 77% in Salmonella typhimurium TA98 and from 0.8 to 99% in strain TA100. There was a direct correlation between the presence of antioxidant activity and antimutagenic activity of the plant extracts. Although no plant extract had mutagenic activity on its own, some of the plant extracts enhanced the mutagenicity of 4-NQO, a phenomenon referred to as comutagenicity.ConclusionsSome of the plant extracts investigated in this study had potential antimutagenic activities. The antimutagenic activities may be associated with the presence of antioxidant polyphenols in the extracts. From the results plant extracts were identified that were not mutagenic, not cytotoxic and that may be antimutagenic in the Ames test. For most plant extracts, at the highest concentration used (5 mg/ml), the level of antimutagenicity was below the recommended 45% to conclude whether plants have good antimutagenic activity. However, in most screening studies for antimutagenesis, a 20% decrease in the number of revertants must be obtained in order to score the extract as active. Psoralea pinnata L. had the highest percentage antimutagenicity recorded in this study (76.67 and 99.83% in S. typhimurium TA98 and TA100 respectively) at assayed concentration of 5 mg/ml.The results indicate that investigating antioxidant activity and the number of antioxidant compounds in plant extracts could be a viable option in searching for antimutagenic compounds in plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1437-x) contains supplementary material, which is available to authorized users.