Inhibition of doxorubicin-induced mutagenicity by Baccharis dracunculifolia

Baccharis dracunculifolia DC (Asteraceae), a native plant from Brazil, have been used as an antipyretic, stomachic and health tonic in Brazil. The objective of the present study was to investigate the potential mutagenic effect of B. dracunculifolia ethyl acetate extract (Bd-EAE) and its influence on the mutagenicity induced by the chemotherapeutic agent doxorubicin (DXR) using the rat bone marrow and peripheral blood micronucleus test. Wistar rats were divided into 10 treatment groups. Five groups received DXR (90 mg/kg body weight, b.w., intraperitoneally) to induce mutagenicity and three of these groups received a single oral dose of Bd-EAE at a concentration of 6, 12 or 24 mg/kg b.w. prior to DXR administration. A vehicle-treated control group and Bd-EAE control groups were also included. The results showed that Bd-EAE itself was not mutagenic, in the rat micronucleus assay. In animals treated with Bd-EAE and DXR, the number of MNPCEs was significantly decreased compared to animals receiving DXR alone. HPLC analysis of the extract obtained permitted the identification of the following phenolic compounds: caffeic acid, p-coumaric acid, aromadendrin-4'O-methyl ether, 3-prenyl-p-coumaric acid (drupanin), 3,5-diprenyl-p-coumaric acid (artepillin C) and baccharin. The putative antioxidant activity or the interference of one or more of the active compounds of Bd-EAE with mutagenic metabolic pathways may explain its effect on DXR mutagenicity.     

Antimutagenicity of rosmarinic acid in Swiss mice evaluated by the micronucleus assay

Rosmarinic acid (RA) is a natural phenolic compound which presents different biological activities such as antitumor, antibacterial, anti-inflammatory, hepatoprotective and cardioprotective properties. In view of its important biological activities, the study of the effects of RA on genetic material becomes relevant. Thus, the objective of the present study was to evaluate the mutagenic and/or antimutagenic potential of RA on peripheral blood cells of Swiss mice using the micronucleus assay. Three doses of RA (50, 100 and 200 mg/kg body weight, b.w.) were used for the evaluation of its mutagenic potential. In the antimutagenicity assays, the different concentrations of RA were combined with the chemotherapeutic agent doxorubicin (DXR, 15 mg/kg b.w.). Peripheral blood samples were collected 24, 48 and 72 h after treatment for the evaluation of micronucleated polychromatic erythrocytes (MNPCEs). The results of the mutagenicity assays showed no increase in the frequency of micronuclei in animals treated with different concentrations of RA when compared to the negative controls. Treatment with different concentrations of RA combined with DXR revealed a significant reduction in the frequency of micronuclei compared to animals treated with DXR only. Although the mechanisms underlying the protective effect of RA are not completely understood, the putative antioxidant activity of RA might explain its effect on DXR mutagenicity.     

Antimutagenicity of rosmarinic acid in Swiss mice evaluated by the micronucleus assay

Rosmarinic acid (RA) is a natural phenolic compound which presents different biological activities such as antitumor, antibacterial, anti-inflammatory, hepatoprotective and cardioprotective properties. In view of its important biological activities, the study of the effects of RA on genetic material becomes relevant. Thus, the objective of the present study was to evaluate the mutagenic and/or antimutagenic potential of RA on peripheral blood cells of Swiss mice using the micronucleus assay. Three doses of RA (50, 100 and 200 mg/kg body weight, b.w.) were used for the evaluation of its mutagenic potential. In the antimutagenicity assays, the different concentrations of RA were combined with the chemotherapeutic agent doxorubicin (DXR, 15 mg/kg b.w.). Peripheral blood samples were collected 24, 48 and 72 h after treatment for the evaluation of micronucleated polychromatic erythrocytes (MNPCEs). The results of the mutagenicity assays showed no increase in the frequency of micronuclei in animals treated with different concentrations of RA when compared to the negative controls. Treatment with different concentrations of RA combined with DXR revealed a significant reduction in the frequency of micronuclei compared to animals treated with DXR only. Although the mechanisms underlying the protective effect of RA are not completely understood, the putative antioxidant activity of RA might explain its effect on DXR mutagenicity.     

Fungal biotransformation of p-coumaric acid into caffeic acid by Pycnoporus cinnabarinus: an alternative for producing a strong natural antioxidant

Fungal biotransformation of p-coumaric acid into caffeic acid, potentially a strong antioxidant, was evidenced in Pycnoporus cinnabarinus cultures grown with high feeding of p-coumaric acid. Preliminary experiments showed no toxicity of both p-coumaric and caffeic acids at concentrations ranging from 0 to 500 mg l^–1. Feeding 450 mg p-coumaric acid l^–1 into P. cinnabarinus cultures grown on 20 g l^–1 glucose medium resulted in the production of 257 mg caffeic acid l^–1with a molar yield of 21%.     

Evaluation of the mutagenicity and antimutagenicity of Ziziphus joazeiro Mart. bark in the micronucleus assay

The aim of this study was to evaluate the mutagenicity (clastogenicity/aneugenicity) of a glycolic extract of Ziziphus joazeiro bark (GEZJ) by the micronucleus assay in mice bone marrow. Antimutagenic activity was also assessed using treatments associated with GEZJ and doxorubicin (DXR). Mice were evaluated 24–48 h after exposure to positive (N-nitroso-N-ethylurea, NEU - 50 mg.kg⁻¹ and DXR - 5 mg.kg⁻¹) and negative (150 mM NaCl) controls, as well as treatment with GEZJ (0.5–2 g.kg⁻¹), GEZJ (2 g.kg⁻¹) + NEU and GEZJ (2 g.kg⁻¹) + DXR. There were no significant differences in the frequencies of micronucleated polychromatic erythrocytes in mice treated with GEJZ and GEJZ + DXR compared to the negative controls, indicating that GEZJ was not mutagenic. Analysis of the polychromatic:normochromatic erythrocyte ratio revealed significant differences in the responses to doses of 0.5 g.kg⁻¹ and 1–2 g.kg⁻¹ and the positive control (NEU). These results indicated no systemic toxicity and moderate toxicity at lower and higher doses of GEZJ. The lack of mutagenicity and systemic toxicity in the antimutagenic assays, especially for treatment with GEZJ + DXR, suggested that phytochemical compounds in Z. joazeiro bark attenuated DXR-induced mutagenicity and the moderate systemic toxicity of a high dose of Z. joazeiro bark (2 g.kg⁻¹). Further studies on the genotoxicity of Z. joazeiro extracts are necessary to establish the possible health risk in humans and to determine the potential as a chemopreventive agent for therapeutic use.     

Antimutagenicity of ursolic acid and oleanolic acid against doxorubicin-induced clastogenesis in Balb/c mice

Ursolic acid (UA) and oleanolic acid (OA) are triterpenoid compounds found in food, medicinal herbs and various other plants in free form or bound to glycosides. Both substances are known for their antimicrobial, hepatoprotective, anti-inflammatory, antiallergic, antiviral and cytotoxic activities. In the present study, we evaluated the antimutagenic potential of UA and OA using the micronucleus test in peripheral blood and bone marrow of Balb/c mice. The animals were divided into 10 treatment groups: mice treated with UA (80 mg/kg b.w.); OA (80 mg/kg b.w.); a mixture of UA and OA (80 mg/kg b.w.); the antineoplastic agent doxorubicin (DXR, 90 mg/kg b.w.); DMSO and DXR; UA and DXR; OA and DXR; UA, OA and DXR, and negative and solvent controls. UA, OA and a mixture of UA and OA were administered to the animals by gavage, followed by the intraperitoneal injection of DXR. The results showed a significant reduction in micronucleus frequency in the groups concomitantly treated with the triterpenoid compounds and DXR compared to that treated with DXR alone. The present results demonstrate the antimutagenic activity of UA and OA under the experimental conditions used in this study.     

p-香豆酸对西洋参的化感作用及生理机制

西洋参(Panax quinquefolium L.)栽培中存在严重的连作障碍现象,前期发现p-香豆酸在以滤纸片为基质的条件下,能够显著抑制西洋参胚根的生长。为了明确p-香豆酸在土壤基质中对种胚的化感活性以及对成株西洋参生长的作用及生理机制,以自然土壤为基质,观察p-香豆酸作用后种胚的生长情况;采用室内水培试验,观察p-香豆酸作用下2年生西洋参种根从出苗至结果期的生长及部分生理指标的变化。种胚生长实验在土壤中分别添加0.0024、0.012、0.06、0.3、1.5、7.5 mg/g的p-香豆酸,处理7 d后测定西洋参种胚的胚根长和胚芽长。水培试验中全营养液中分别添加0.012 mg/mL、0.06 mg/mL、0.3 mg/mL p-香豆酸,处理后每隔5 d测定植株叶片展开情况、株高、冠幅等生长指标;于展叶期(10 d)、现蕾期(20 d)、结果期(30 d)测定地上部分及新生须根的生物量,同时测定新生须根苯丙氨酸解氨酶(PAL)活力;叶片完全展开后测定植株净光合速率(Pn)、表观电子传递速率(ETR)和最大光化学效率(Fv/Fm)等光合特性参数。结果表明,土壤中添加0.0024-7.5 mg/g p-香豆酸西洋参胚根长度降低28.52%-100%,胚芽长度降低1.09%-100%,并呈现一定的剂量抑制效应。实验浓度内的p-香豆酸可显著抑制西洋参植株地上部分生长,推迟展叶期;结果期地上部生物量比对照降低17.17%-54.55%(P < 0.05,Dunnett-t test);同时,叶片的Pn、ETR受到抑制(P < 0.05),但Fv/Fm不变;对须根的影响主要表现为0.06 mg/mL p-香豆酸处理组在展叶期PAL酶活力提高69.05%,之后PAL活力和生物量均比对照下降,浓度增加至0.3 mg/mL时整个培养期内PAL酶活力和生物量均低于对照。由此推论,根系环境中的p-香豆酸在自然土壤中对西洋参种胚具有显著抑制其生长的化感作用;对成株西洋参的作用主要为抑制地上部分生长,并通过降低成株西洋参叶片光合能力,从而表现出明显的化感作用,0.06 mg/mL p-香豆酸诱导须根PAL酶活力先升高再降低并最终降低生物量的结果也表明p-香豆酸是西洋参根系生长的胁迫因素。结果证实p-香豆酸对西洋参种胚和成株的生长均具有自毒作用,其抑制生长的生理机制在于抑制叶片的光合作用。     

p-Coumaric acid — a monomer in the sporopollenin skeleton

Sporopollenin obtained from wings of Pinus mugo (Turra) pollen was analysed by pyrolysis mass spectrometry. In the spectrum, mass peaks which are characteristic for p-coumaric acid were dominant. p-Coumaric acid was the main degradation compound when the wing material was treated by a gentle method using AII₃, and also when the remaining residue of the treated sporopollenin material was saponified. It is therefore assumed that p-coumaric acid is a genuine structural unit in the sporopollenin skeleton. In addition, the effects of AII₃ treatment indicate that the p-coumaric acid might be bound by ether linkages.Abbreviations HPLC high-performance liquid chromatography - MS mass spectrometry - TLC thin-layer chromatography     

Mutagenicity and antimutagenicity of <Emphasis Type="Italic">Croton cajucara</Emphasis>

Croton cajucara Benth. (‘sacaca’) is a tree of the Euphorbiaceae family, native to the Amazon region in northern Brazil, where it is widely used in the popular treatment of various diseases. Its active principle, the terpenoid trans-dehydrocrotonin, has been credited with a variety of medical properties, including antiulcer, antiinflammatory, antitumor, antimutagenic and hypoglycemic activity. In this investigation, possible mutagenic and antimutagenic effects were evaluated in treatments using methanol extract of this plant on Swiss Albino mice by examining their peripheral blood cells for micronuclei. In these tests, the material obtained by methanol extraction of C. cajucara tree bark was administered to the mice by gavage. None of the doses evaluated in this study presented mutagenicity. Analysis of the results obtained from studies evaluating antimutagenicity revealed protection against the chemotherapeutic agent cyclophosphamide for the two highest doses used.     

The protective effect exerted by ascorbic acid on DNA fragmentation of human leukocytes induced by Lachesis muta muta venom

The objective of this study was to evaluate the genotoxic and mutagenic effects of the toxins present in Lachesis muta muta's venom on human peripheral blood leukocytes and the protective potential of ascorbic acid on DNA fragmentation. The venom of L. muta muta was incubated in different concentrations (1, 2.5, 5, 7.5, 10, 15, 20, 30, 40, 50, 60, and 120 µg/mL) with human blood to evaluate DNA fragmentation using the comet, agarose gel electrophoresis, and micronucleus assays. In these concentrations evaluated, the venom of L. muta muta induced genotoxicity (comet assay and agarose gel electrophoresis) and mutagenicity (micronucleus test), but they were not cytotoxic, as they did not change the rate of cell proliferation after cytokinesis blockade with cytochalasin B. The ascorbic acid significantly inhibited the genotoxicity induced by L. muta muta venom in the proportions evaluated (1:0.1 and 1:0.5, venom/ascorbic acid - w/w). Thus, future studies are needed to elucidate the protective mechanisms of ascorbic acid on the genotoxic effects induced by toxins present in snake venoms.     

Oxidation of hydroxycinnamic acid and hydroxycinnamyl alcohol derivatives by laccase and peroxidase. Interactions among p-hydroxyphenyl,guaiacyl and syringyl groups during the oxidation reactions

Fungal laccase oxidized derivatives of hydroxycinnamic acid. The rates decreased in the order sinapic acid > ferulic acid ≥p-coumaric acid. The laccase oxidized sinapyl alcohol faster than coniferyl alcohol. The rates of oxidation of the hydroxycinnamic acid derivatives by an isoenzyme of peroxidase from horseradish decreased in the order p-coumaric acid > ferulic acid ≥ sinapic acid. The peroxidase oxidized coniferyl alcohol much faster than sinapyl alcohol. The laccase and the peroxidase predominantly oxidized (a) ferulic acid in a reaction mixture that contained p-coumaric acid and ferulic acid, (b) sinapic acid in a mixture of p-coumaric acid plus sinapic acid, and (c) sinapic acid in a mixture of ferulic acid plus sinapic acid. In a reaction mixture that contained both coniferyl and sinapyl alcohols, both fungal laccase and horseradish peroxidase predominantly oxidized sinapyl alcohol. From these results, it is concluded (1) that the p-hydroxyphenyl radical can oxidize guaiacyl and syringyl groups and produce their radicals and (2) that the guaiacyl radical can oxidize the syringyl group under formation of its radical; and that (3) in both cases the reverse reactions are very slow.     

Grape skins (Vitis vinifera L.) catalyze the in?vitro enzymatic hydroxylation of p-coumaric acid to caffeic acid

The ability of grape skins to catalyze in vitro conversion of p-coumaric acid to the more potent antioxidant caffeic acid was studied. Addition of different concentrations of p-coumaric to red grape skins (Cabernet Sauvignon) resulted in formation of caffeic acid. This caffeic acid formation (Y) correlated positively and linearly to p-coumaric acid consumption (X): Y = 0.5 X + 9.5; R ² = 0.96, P < 0.0001. The kinetics of caffeic acid formation with time in response to initial p-coumaric acid levels and at different grape skin concentrations, indicated that the grape skins harboured an o-hydroxylation activity, proposedly a monophenol- or a flavonoid 3′-monooxygenase activity (EC 1.14.18.1 or EC 1.14.13.21). The K _m of this crude o-hydroxylation activity in the red grape skin was 0.5 mM with p-coumaric acid.     

Is There Any Possible Genotoxic Effect in Exfoliated Bladder Cells of Rat Under the Exposure of 1800 MHz GSM-Like Modulated Radio Frequency Radiation (RFR)?

People are exposed to many carcinogenic and mutagenic chemicals in their everyday lives. These include antineoplastic drugs, Polycyclic aromatic hydrocarbons (PAH)s, aromatic amines, nitrosamines, metals, and electromagnetic radiation. Based on the state of knowledge acquired during the last 50 years of research on possible biological effects of electromagnetic fields (EMF), the majority of the scientific community is convinced that exposure to EMF below the existing security limits does not cause a risk to the health of the general public. However, this position is questioned by others, who are of the opinion that the available research data are contradictory or inconsistent and, therefore, unreliable.

In this study, we aimed to investigate if there is any effect of 1800?MHz GSM modulated radio frequency radiation (RFR) on the number of micronucleus in exfoliated bladder cells of rat which will be informative about the genotoxic damage. Exposure period was 20?min/day, 5 days/week during a month. Six female Wistar rats were used for two groups: Group I (n=6): controls; Group II (n=6): 1.8?GHz exposed animals. 1800?MHz RFR did not showed a significant MN frequencies in rat bladder cells when compared with the control group (p>0.05). 1800?MHz RFR-exposed animals did not produce any genotoxic effect when compared with the control group ( p>0.05). Kinetic studies are important for any biomarker, especially those in which tissue differentiation and maturation processes will heavily influence the time between induction of damage and collection of damaged cells for micronucleus analysis.     

Lipid Accumulation during Canola Seed Germination in Response to Cinnamic Acid Derivatives

The objective of this research was to investigate how ferulic and p-coumaric acids affect lipid and fatty acid composition during canola (Brassica napus L.) seed germination. Data showed that both compounds increased total lipid and fatty acid contents in the cotyledons during germination. The largest accumulation in lipids occurred at 1.0 mM p-coumaric acid with an increase in all unsaturated fatty acids. The results suggest that allelochemicals interfere in canola seed germination by reducing lipid mobilization.     

Stimulation of indoleacetic acid production in a <Emphasis Type="Italic">Rhizobium</Emphasis> isolate of <Emphasis Type="Italic">Vigna mungo</Emphasis> by root nodule phenolic acids

The influence of endogenous root nodules phenolic acids on indoleacetic acid (IAA) production by its symbiont (Rhizobium) was examined. The root nodules contain higher amount of IAA and phenolic acids than non-nodulated roots. Presence of IAA metabolizing enzymes, IAA oxidase, peroxidase, and polyphenol oxidase indicate the metabolism of IAA in the nodules and roots. Three most abundant endogenous root nodule phenolic acids (protocatechuic acid, 4-hydroxybenzaldehyde and p-coumaric acid) have been identified and their effects on IAA production by the symbiont have been studied in l-tryptophan supplemented yeast extract basal medium. Protocatechuic acid (1.5 μg ml⁻¹) showed maximum stimulation (2.15-fold over control) of IAA production in rhizobial culture. These results indicate that the phenolic acids present in the nodule might serve as a stimulator for IAA production by the symbiont (Rhizobium). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Enhanced transformation of malachite green by laccase of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> in the presence of natural phenolic compounds

In this study, we investigated the efficacy of phenolic extract of wheat bran and lignin-related phenolic compounds as natural redox mediators on laccase-mediated transformation of malachite green (MG) using purified laccase from the white-rot fungus Ganoderma lucidum. G. lucidum laccase was able to decolorize 40.7% MG dye (at 25 mg l⁻¹) after 24 h of incubation. Whereas, the addition of phenolic extract of wheat bran enhanced the decolorization significantly (p < 0.001) by two- to threefold than that of purified laccase alone. Among various natural phenolic compounds, acetovanillone, p-coumaric acid, ferulic acid, syringaldehyde, and vanillin were the most efficient mediators, as effective as the synthetic mediator 1-hydroxybenzotriazole. Characterization of MG transformation products by HPLC, UV–Vis, and liquid chromatography-mass spectrometry-electrospray ionization analysis revealed that N-demethylation was the key mechanism of decolorization of MG by laccase. Growth inhibition test based on mycelial growth inhibition of white rot fungus Phanerochaete chrysosporium revealed that treatment with laccase plus natural mediators effectively reduced the growth inhibitory levels of MG than that of untreated one. Among all the tested compounds, syringaldehyde showed the highest enhanced decolorization, as a consequence reduced growth inhibition was observed in syringaldehyde-treated samples. The results of the present study revealed that the natural phenolic compounds could alternatively be used as potential redox mediators for effective laccase-mediated decolorization of MG.     

Mutagenic activity of the glutathione S-transferase substrate 1-chloro-2,4-dinitrobenzene (CDNB) in the Salmonella mutagenicity assay

The mutagenicity of the commonly used glutathione S-transferase substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) was investigated in the Salmonella mutagenicity assay. CDNB induced a concentration-dependent mutagenic response in Salmonella typhimurium strain TA98. Incorporation of an activation system derived from Aroclor 1254-induced rats did not influence mutagenic response. Under the same conditions DCNB failed to display mutagenic activity. The mutagenic activity of CDNB was attenuated in bacterial strains under-expressing nitroreductase or O-acetylase activity but, in contrast, it was exaggerated in an O-acetylase over-expressing strain. It is inferred that CDNB exhibits a mutagenic response following reduction of the nitro-group to the hydroxylamine, which is further acetylated to form the acetoxy derivative that presumably breaks down spontaneously to generate the nitrenium ion, the likely ultimate mutagen.     

Biosynthesis of p-hydroxybenzoic acid in elicitor-treated carrot cell cultures

Carrot (Daucus carota L.) cells respond to treatment with fungal elicitors by synthesizing wallbound p-hydroxybenzoic acid (p-HBA). The biosynthetic pathway to p-HBA is still hypothetical. Tracer experiments with l-phenylalanine indicate the involvement of the general phenylpropanoid pathway. 3,4 (Methylenedioxy) innamic acid, an inhibitor of hydrocycinnamate CoA ligase, inhibits the accumulation of anthocyanins in carrot, while it does not interfere with p-HBA synthesis. Thus p-HBA biosynthesis does not appear to involve CoA thioesters. In the present report the sequence of enzymic reactions leading to p-HBA was investigated in vitro using protein preparations from cells treated with a fungal elicitor from Pythium aphanidermatum (Edson) Fitzp. The side-chain degradation from p-coumaric acid to p-HBA is not analogous to the -oxidation of fatty acids and involves p-hydroxybenzaldehyde as an intermediate. The final step from p-hydroxybenzaldehyde to p-HBA is catalyzed by an NAD-dependent p-hydroxybenzaldehyde dehydrogenase (EC 1.2.1.-). This reaction was characterized with regard to cofactor requirements, pH and temperature optima. The in-vitro formation of p-HBA from p-coumaric acid and the activity of the hydroxybenzaldehyde dehydrogenase are moderately elicitor-induced but to a much lesser extent than phenylalanine ammonialyase, which is the starting enzyme of the general phenylpropanoid pathway.Abbreviations HPLC high-performance liquid chromatography - MDCA 3,4-(methylenedioxy)-cinnamic acid - p-HBA p-hydroxybenzoic acid This work was supported by a grant from the Deutsche Forschungsgemeinschaft and a sholarship of the Land Baden-Württemberg (J.-P. S.).     

Rapid and simple assay for feruloyl and p-coumaroyl esterases

A rapid, simple and sensitive method for detection of ferulic and p-coumaric acids using HPLC has been developed which can be used to determine the respective phenolic acid esterase activities of microorganisms. Prior concentration, purification or derivatization of the samples are not required. As little as 0.5 mg ferulic or p-coumaric acid/I could be detected and estimated in < 1 h. The method is specific for the two phenolic acids sice no interference by other components was observed.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State (UOFS), PO Box 339, Bloemfontein 93000, South Africa