Antigenotoxicity of artepillin C in vivo evaluated by the micronucleus and comet assays

Artepillin C (3,5‐diprenyl‐p‐coumaric acid), a major compound found in Brazilian green propolis and Baccharis dracunculifolia, shows anti‐inflammatory, antibacterial, antiviral, antioxidant and antitumoral activities, among others. The aim of this study was to evaluate the genotoxic potential of artepillin C and its ability to prevent the chemically induced chromosome breakage or loss and the primary DNA damage using the micronucleus and comet assays in male Swiss mice, respectively. The animals were treated by gavage with different doses of artepillin C (0.4, 0.8 and 1.6 mg kg^?1 b.w.). For the antigenotoxicity assays, the different doses of artepillin C were administered simultaneously to doxorubicin (DXR; micronucleus test; 15 mg kg^?1 b.w.) and to methyl methanesulfonate (MMS; comet assay; 40 mg kg^?1 b.w.). The results showed that artepillin C itself was not genotoxic in the mouse micronucleus and comet assays. In the animals treated with artepillin C and DXR, the number of micronucleated reticulocytes was significantly lower in comparison with the animals treated only with DXR. Regarding antigenotoxicity, artepillin C at the tested doses significantly reduced the extent of DNA damage in liver cells induced by MMS. Copyright © 2011 John Wiley & Sons, Ltd.     

In vitro cytotoxicity,genotoxicity and antigenotoxicity assessment of Solanum lycocarpum hydroalcoholic extract

Context: Solanum lycocarpum A. St.-Hil. (Solanaceae), popularly known as ‘fruta-do-lobo’ (wolf fruit), ‘lobeira’ and ‘jurubebão’, is commonly used by native people of Central Brazil in powder form or as a hydroalcoholic extract for the management of diabetes and obesity and to decrease cholesterol levels. Objective: The present study determines the possible cytotoxic, genotoxic and antigenotoxic activities of hydroalcoholic extract of the S. lycocarpum fruits (SL).

Materials and methods: The clonogenic efficiency assay was used to determine the cytotoxicity. Three concentrations of SL (16, 32 and 64?μg/mL) were used for the evaluation of its genotoxic and antigenotoxic potential on V79 cells using the micronucleus and comet assays. In the antigenotoxicity assays, the cells were treated simultaneously with SL and the alkylating agent methyl methanesulphonate (MMS, 44?μg/mL for the micronucleus assay and 22?μg/mL for the comet assay) as an inducer of micronuclei and DNA damage.

Results: The results showed that SL was cytotoxic at concentrations up to 64?μg/mL. No significant differences in the rate of chromosome or DNA damage were observed between cultures treated with SL and the control group. In addition, the frequencies of micronuclei and DNA damage induced by MMS were significantly reduced after treatment with SL. The damage reduction percentage ranged from 68.1% to 79.2% and 12.1% to 16.5% for micronucleus and comet assays, respectively.

Discussion and conclusion: SL exerted no genotoxic effect and exhibited chemopreventive activity against both genomic and chromosome damage induced by MMS.     

Protective effects of Scutellaria lindbergii root extract against oxidative-induced cell and DNA damage in mouse fibroblast-like cells

Context: Scutellaria lindbergii Rech. f. (Lamiaceae) is an Iranian species of Scutellaria which has been shown to exert antimicrobial, antioxidant and cytotoxic effects. Objective: The protective properties of total methanol extract (TME) of S. lindbergii and its fractions (defatted and CH₂Cl₂) were investigated against cytotoxic and genotoxic effects of H₂O₂ in NIH 3T3 cell line as non-malignant cells. Materials and methods: The cells were incubated with different concentrations of S. lindbergii root extracts [TME (15–250?μg ml^?1), defatted fraction (15–500?μg ml^?1) and CH₂Cl₂ fraction (5–40?μg ml^?1)] and toxic concentration of H₂O₂ (200?µM) at 37?°C for 2?h concurrently and Cell viability was quantitated by MTT assay. The antigenotoxic effect of extracts was investigated using comet assay. The cells were incubated with extracts [TME (25–250?μg ml^?1), defatted fraction (25–500?μg ml^?1) and CH₂Cl₂ fraction (5–40?μg ml^?1)] and H₂O₂ (25?µM) at 4?°C for 20?min, then the comet assay was performed. DNA damage was expressed as percentage tail DNA. Results: Total methanol extract of S. lindbergii and its fractions had a significant inhibitory effect on DNA damage. The IC₅₀ values of TME, defatted fraction and CH₂Cl₂ fraction against DNA damage were determined as 48, 138 and 8?μg ml^?1, respectively. Conclusion: S. lindbergii extracts can prevent oxidative DNA damage, which is likely due to its flavonoids and phenolic compounds as antioxidant constituents.     

Effect of green juice and their bioactive compounds on genotoxicity induced by alkylating agents in mice

ABSTRACT

Kale juice (Brassica oleracea L. var. acephala D.C.) is a reliable source of dietary carotenoids and typically contains the highest concentrations of lutein (LT) and beta-carotene (BC) among green leafy vegetables. As a result of their antioxidant properties, dietary carotenoids are postulated to decrease the risk of disease occurrence, particularly certain cancers. The present study aimed to (1) examine the genotoxic and antigenotoxic activity of natural and commercially available juices derived from Brassica oleracea and (2) assess influence of LT or BC against DNA damage induced by alkylating agents such as methyl methanesulfonate (MS) or cyclophosphamide (CP) in vivo in mice. Male Swiss mice were divided into groups of 6 animals, which were treated with water, natural, or commercial Brassica oleraceae juices (kale), LT, BC, MMS, or CP. After treatment, DNA damage was determined in peripheral blood lymphocytes using the comet assay. Results demonstrated that none of the Brassica oleraceae juices or carotenoids produced genotoxic effects. In all examined cell types, kale juices or carotenoids inhibited DNA damage induced by MMS or CP administered either pre- or posttreatment by 50 and 20%, respectively. Under our experimental conditions, kale leaf juices alone exerted no marked genotoxic or clastogenic effects. However, a significant decrease in DNA damage induced by MMS or CP was noted. This effect was most pronounced in groups that received juices, rather than carotenoids, suggesting that the synergy among constituents present in the food matrix may be more beneficial than the action of single compounds. Data suggest that the antigenotoxic properties of kale juices may be of therapeutic importance.     

In vitro effect of nickel on bovine spermatozoa motility and annexin V‐labeled membrane changes

In this study the effect of in vitro culture of bovine spermatozoa with nickel (NiCl₂) on spermatozoa motility and membrane changes was analyzed. The spermatozoa motility significantly decreased after 120 min of culture at the concentration of 1000 μm Ni ml^?1 (P < 0.05) and after 240 min of culture at the concentration of 500 and 1000 μm Ni ml^?1 (P < 0.001) as compared with control. The progressive motility was the highest in the control group and in the groups with the lowest nickel concentrations (7.8 and 125 μm Ni ml^?1). The progressive spermatozoa motility was significantly altered even after 30 min of culture in the group with the highest nickel concentration (1000 μm Ni ml^?1). A significant decrease in progressive motility from the concentration of 250 μm Ni ml^?1 was detected after 240 min of culture. Concentrations from 125 μm Ni ml^?1 in various time periods of culture stimulated spermatozoa motility after 30 min (P < 0.001), but later an inhibitory effect was noted. After 240 min of in vitro spermatozoa culture with 125 μm Ni ml^?1 a typical Annexin V fluorescence reaction was detected. Fluorescence was detected in mitochondrial segment of bovine spermatozoa. In spermatozoa exposed to higher nickel concentrations the Annexin V‐positive reaction was detected also on the spermatozoa head membrane. In the group with the highest concentration and the longest time of exposure (1000 μm Ni ml^?1; 240 min) the apoptotic Annexin‐positive regions were detected not only in the mitochondrial part, but also in the spermatozoa head (acrosomal and postacrosomal part), showing significant alteration of spermatozoa membrane integrity. Copyright © 2010 John Wiley & Sons, Ltd.     

In vitro genotoxic and antigenotoxic effects of an exopolysaccharide isolated from Lactobacillus salivarius KC27L

Exopolysaccharide isolated from Lactobacillus salivarius (new genus name Ligilactobacillus) KC27L strain (EPS_KC27L) exhibits antioxidant properties with 1,1-diphenyl-2-picrylhydrazase (DPPH) radical and superoxide anion radical (O₂^-.) scavenging effect and iron ion (Fe²⁺) chelating activity. This study aimed to investigate the in vitro genotoxic effects of EPS_KC27L alone (12.50, 25.00, 50.00, and 100.00 μg/mL) and its antigenotoxic activity against DNA damage induced by mitomycin-C (MMC; 0.20 μg/mL), methyl methanesulfonate (MMS; 5.00 μg/mL), and hydrogen peroxide (H₂O₂; 100 μM). For this purpose, chromosome aberration (CA), sister chromatid exchange (SCE), micronucleus (MN), and comet assays were performed in human peripheral lymphocytes. In addition, the structure of EPS_KC27L was investigated in the scanning electron microscope (SEM). EPS_KC27L alone did not cause a significant genotoxic effect in CA, SCE, MN, and comet tests. EPS_KC27L significantly decreased the frequency of CA, SCE, and MN induced by MMC and MMS. EPS_KC27L also significantly reduced DNA damage induced by H₂O₂. This study showed that the EPS_KC27L alone has no genotoxic risk at these concentrations and shows antigenotoxic activity against MMC, MMS, and H₂O₂. Consequently, EPS_KC27L was found to exhibit chemopreventive activity against genotoxic agents. This effect is believed to be due to the antioxidant properties of EPS_KC27L.     

A New Method for the Measurement of Nitrosoureas in Plasma: an H.P.L.C. Procedure for the Measurement of Fotemustine Kinetics

1. An analytical method for a novel nitrosourea, fotemustine, has been developed using solid-phase extraction and?h.p.l.c. with u.v. detection. As part of the development, different methods for stabilising fotemustine after sample collection have been investigated. The method has been successfully applied to pharmacokinetic studies in monkeys and man.

2. Providing plasma was separated immediately from blood and frozen within 3?min of collection, negligible degradation of fotemustine occurred. The samples could then be stored at —20°C in the dark for up to six days particularly if thawing prior to analysis was accelerated using a 50°C water-bath so that it was complete within 3?min. Equivalent results were also obtained with samples stabilised with 0·1?m citric acid immediately after the preparation of plasma.

3. The analytical method showed good precision with a within-day variation ranging between ±10.7% at the lowest concentration investigated (0.1 μg ml^?1) to 2.0% at 50.0 μgml^?1. The accuracy of measurement was from 108.9% to 97.6% at 0.1 and 50.0 μg ml^?1 respectively and the response was linear up to 50 μg ml^?1. The minimum level of quantitation was 20 ng ml^?1.

4. After a single intravenous bolus dose of [¹⁴C]fotemustine (100mg m^?2) to Cynomolgus monkeys, intact drug levels rapidly declined (t_1/2 12.6±0.5?min) although the halflife of radioactivity (approx 100?h) was much longer. The plasma clearance of fotemustine was 225±63 ml min^?1 with a volume of distribution based on area of 4.1±1.2 litres.

5. As with monkey, plasma levels of intact fotemustine in a patient given [¹⁴C]-drug as a 1?h constant rate intravenous infusion (approx. 100?mg m^?2), declined rapidly but with a half-life of 23.2?min. Again, the half-life for total radioactivity was considerably longer (30.8?h). The plasma clearance was 1426 ml min^?1 and the volume of distribution based on area was 47.71.     

Multi‐walled carbon nanotubes induce cytotoxicity and genotoxicity in human lung epithelial cells

The increasing use of nanomaterials in consumer products highlights the importance of understanding their potential toxic effects. We evaluated cytotoxic and genotoxic/oxidative effects induced by commercial multi‐walled carbon nanotubes (MWCNTs) on human lung epithelial (A549) cells treated with 5, 10, 40 and 100 µg ml^?1 for different exposure times. Scanning electron microscopy (SEM) analysis, MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide] and lactate dehydrogenase (LDH) assays were performed to evaluate cytotoxicity. Fpg‐modified comet assay was used to evaluate direct‐oxidative DNA damage. LDH leakage was detected after 2, 4 and 24 h of exposure and viability reduction was revealed after 24 h. SEM analysis, performed after 4 and 24 h exposure, showed cell surface changes such as lower microvilli density, microvilli structure modifications and the presence of holes in plasma membrane. We found an induction of direct DNA damage after each exposure time and at all concentrations, statistically significant at 10 and 40 µg ml^?1 after 2 h, at 5, 10, 100 µg ml^?1 after 4 h and at 10 µg ml^?1 after 24 h exposure. However, oxidative DNA damage was not found. The results showed an induction of early cytotoxic effects such as loss of membrane integrity, surface morphological changes and MWCNT agglomerate entrance at all concentrations. We also demonstrated the ability of MWCNTs to induce early genotoxicity. This study emphasizes the suitability of our approach to evaluating simultaneously the early response of the cell membrane and DNA to different MWCNT concentrations and exposure times in cells of target organ. The findings contribute to elucidation of the mechanism by which MWCNTs cause toxic effects in an in vitro experimental model. Copyright © 2012 John Wiley & Sons, Ltd.     

Purified c‐phycoerythrin: safety studies in rats and protective role against permanganate‐mediated fibroblast‐DNA damage

We have evaluated in vitro cytotoxicity of cyanobacterial phycoerythrin (C‐PE) on three human cell lines by cell proliferation and neutral red uptake assays. No toxic effects of C‐PE were observed to any of the cell lines tested. The protective role of purified C‐PE to potassium permanganate‐mediated human fibroblast‐DNA damage was assessed by comet assay at 0 (control), 10 and 20 µg C‐PE ml^?1 doses in pre‐, simultaneous and post‐mutagen exposure conditions. Significant DNA damage was detected only in post‐mutagen exposure conditions. Our findings confirmed that the C‐PE is non‐toxic and provides protection against permanganate‐mediated DNA damage. The preliminary acute (2000 mg C‐PE kg^?1 body weight, b.w.) and 90 day sub‐chronic (0, 5, 15 and 25 mg C‐PE kg^?1 b.w./day) oral toxicity studies of purified C‐PE in male albino rats showed no mortality or treatment‐related major clinical signs, and all the doses of C‐PE were well tolerated. The no observed adverse effect level and no observed effect level were found to be 15 and 5 mg C‐PE kg^?1 b.w./day respectively. Copyright © 2010 John Wiley & Sons, Ltd.     

Lycopene activity against chemically induced DNA damage in Chinese hamster ovary cells.

Lycopene is a natural pigment synthesized by plants and microorganisms, and it is mainly found in tomatoes. It is an acyclic isomer of beta-carotene and one of the most potent antioxidants. Several studies have demonstrated the ability of lycopene to prevent chemically induced DNA damage; however, the mechanisms involved are still not clear. In the present study, we investigated the antigenotoxic/antimutagenic effects of lycopene in Chinese Hamster Ovary Cells (CHO) treated with hydrogen peroxide, methylmethanesulphonate (MMS), or 4-nitroquinoline-1-oxide (4-NQO). Lycopene (97%), at final concentrations of 10, 25, and 50 microM, was tested under three different protocols: before, simultaneously, and after the treatment with the mutagens. Comet and cytokinesis-block micronucleus assays were used to evaluate the level of DNA damage. Data showed that lycopene reduced the frequency of micronucleated cells induced by the three mutagens. However, this chemopreventive activity was dependent on the concentrations and treatment schedules used. Similar results were observed in the comet assay, although some enhancements of primary DNA damage were detected when the carotenoid was administered after the mutagens. In conclusion, our findings confirmed the chemopreventive activity of lycopene, and showed that this effect occurs under different mechanisms.     

In vitro evaluation of the cytotoxic and genotoxic effects of artemether,an antimalarial drug,in a gastric cancer cell line (PG100)

Artemisinin is a sesquiterpene lactone endoperoxide, obtained from Artemisia annua, and extensively used as an antimalarial drug. Many studies have reported the genotoxic and cytotoxic effects of artemisinins; however, there are no studies that compare such effects between cancer cell lines and normal human cells after treatment with artemether, an artemisinin derivative. Gastric cancer is the fourth most frequent type of cancer and the second highest cause of cancer mortality worldwide. Thus, the aim of this study was to evaluate the in vitro genotoxic and cytotoxic effects induced by artemether in gastric cancer cell line (PG100) and compare them with the results obtained in human lymphocytes exposed to the same conditions. We used MTT (3‐(4,5‐methylthiazol‐2‐yl)‐2, 5‐diphenyl‐tetrazolium bromide) assay, comet assay and ethidium bromide/acridine orange viability staining to evaluate the cytotoxic and genotoxic effects of artemether in PG100. MTT assay showed a decrease in the survival percentages for both cell types treated with different concentrations of artemether (P < 0.05). PG100 also showed a significant dose‐dependent increase in DNA damage index at concentrations of 119.4 and 238.8 µg ml^?1 (P < 0.05). Our results showed that artemether induced necrosis in PG100 at concentrations of 238.8 and 477.6 µg ml^?1, for all the tested harvest times (P < 0.05). In lymphocytes, artemether induced both apoptosis and necrosis at concentrations of 238.8 and 477.6 µg ml^?1, for all the tested harvest times (P < 0.05). In conclusion, human lymphocytes were more sensitive to the cytotoxic effects of the antimalarial drug than the gastric cancer cell line PG100. Copyright © 2011 John Wiley & Sons, Ltd.     

Amifostine protection against induced DNA damage in γ‐irradiated Escherichia coli cells depend on recN DNA repair gene product activity

Amifostine is the most effective radioprotector known and the only one accepted for clinical use in cancer radiotherapy. In this work, the antigenotoxic effect of amifostine against γ‐rays was studied in Escherichia coli cells deficient in DNA damage repair activities. Assays of irradiated cells treated with amifostine showed that the drug reduced the genotoxicity induced by radiation in E. coli wild‐type genotypes and in uvr, recF, recB, recB‐recC‐recF mutant strains, but not in recN defective cells. Thus, the mechanism of DNA protection by amifostine against γ‐radiation‐induced genotoxicity appears to involve participation of the RecN protein that facilitates repair of DNA double‐strand breaks. The results are discussed in relation to amifostine's chemopreventive potential. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

3‐(3,4‐Dihydroxyphenyl)adenine,a urinary DNA adduct formed in mice exposed to high concentrations of benzene

Metabolism of benzene, an important environmental and industrial carcinogen, produces three electrophilic intermediates, namely, benzene oxide and 1,2‐ and 1,4‐benzoquinone, capable of reacting with the DNA. Numerous DNA adducts formed by these metabolites in vitro have been reported in the literature, but only one of them was hitherto identified in vivo. In a search for urinary DNA adducts, specific LC‐ESI‐MS methods have been developed for the determination in urine of six nucleobase adducts, namely, 7‐phenylguanine, 3‐phenyladenine, 3‐hydroxy‐3,N⁴‐benzethenocytosine, N²‐(4‐hydroxyphenyl)guanine, 7‐(3,4‐dihydroxyphenyl)guanine and 3‐(3,4‐dihydroxyphenyl)‐adenine (DHPA), with detection limits of 200, 10, 260, 50, 400 and 200 pg ml^?1, respectively. Mice were exposed to benzene vapors at concentrations of 900 and 1800 mg m^?3, 6 h per day for 15 consecutive days. The only adduct detected in their urine was DHPA. It was found in eight out of 30 urine samples from the high‐exposure group at concentrations of 352 ± 146 pg ml^?1 (mean ± SD; n = 8), whereas urines from the low‐exposure group were negative. Assuming the DHPA concentration in the negative samples to be half of the detection limit, conversion of benzene to DHPA was estimated to 2.2 × 10^?6% of the absorbed dose. Thus, despite the known high mutagenic and carcinogenic potential of benzene, only traces of a single DNA adduct in urine were detected. In conclusion, DHPA is an easily depurinating adduct, thus allowing indication of only high recent exposure to benzene, but not long‐term damage to DNA in tissues. Copyright © 2012 John Wiley & Sons, Ltd.     

Nonlinear elimination and hepatic concentration of conjugation- metabolite of valproate in guinea-pigs

The plasma clearance and metabolic rate characteristics of valproic acid (VPA) were studied using guinea-pigs placed on various (0.08-9 μmol ml^?1 = 11–1303 μg ml^?1) steady-state plasma concentrations (C_ss) by constant intravenous (i.v.) infusion. The total clearance (CL) was significantly decreased at plasma concentration of 0.61 μmol ml^?1 (88 μg ml^?1). The metabolic clearance of VPA was apparently biphasic. The maximum metabolic rate (V_max) and the Michaelis-Menten constant (K_m) for the primary (V_maxl, K_ml) and the secondary (V_max2, K_m2) pathways were V_maxl = 1.52 μmol min ^?1kg^?1, K_ml = 0.15 μmol ml^?1, V_max2 = 24.98 μmol min ^?1 kg^?1 and K_m2 = 11.70 μmol ml^?1, respectively. The K_ml value was within clinical therapeutic concentration range. The formation of conjugated VPA (cjVPA) metabolite in liver was shown to be saturable. Plasma protein binding of VPA was also nonlinear. The dose-dependent decrease in metabolic clearance was counterbalanced by the increased unbound fraction (f_u), resulting in a relatively constant apparent clearance of VPA over a wide concentration range. The hepatic concentration of VPA was not significantly different from the plasma unbound concentration, again over a wide concentration range. The biliary and hepatic concentrations of VPA were not significantly different; but the concentration ratio of cjVPA in bile compared with that of VPA in liver decreased against hepatic concentration of VPA, which suggests a saturable conjugation rate. The K_m value estimated from hepatic cjVPA production as a function of plasma VPA concentration was comparable with the K_ml value. These results implied that the primary metabolic parameters may describe the conjugation pathway which is nonlinear within the clinical therapeutic concentration range.     

Evaluation of the genotoxicity/mutagenicity and antigenotoxicity/antimutagenicity induced by propolis and Baccharis dracunculifolia,by in vitro study with HTC cells

The ethanolic extract of propolis, especially the Brazilian green type, is widely and mainly used for therapeutic purposes despite the lack of knowledge about its effects and its cellular mode of action. This type of propolis, derived from Baccharis dracunculifolia (alecrim-do-campo), has been extensively commercialized and the consumers use it to enhance health. This work aimed to assess the genotoxic/mutagenic and antigenotoxic/antimutagenic potentials of the ethanolic extracts of Brazilian green propolis and of B. dracunculifolia, on mammalian cells. It was not observed genotoxic and mutagenic effects by both extracts. After evaluate the exposure of the cells to each extract with a recognized mutagen, simultaneously, the results showed a significant reduction on DNA damage. The experiment carried out with a pre-incubation period was more effective than without incubation test, showing that the tested extracts were able to inactivate the mutagen before it could react with the DNA.     

Protective effects of lemongrass (Cymbopogon citratus STAPF) essential oil on DNA damage and carcinogenesis in female Balb/C mice

This study investigated the protective effect of oral treatment with lemongrass (Cymbopogon citratus STAPF) essential oil (LGEO) on leukocyte DNA damage induced by N‐methyl‐N‐nitrosurea (MNU). Also, the anticarcinogenic activity of LGEO was investigated in a multi‐organ carcinogenesis bioassay induced by 7,12‐dimethylbenz(a)antracene, 1,2‐dimethylhydrazine and N‐butyl‐N‐(4‐hydroxibuthyl)nitrosamine in Balb/C female Balb/c mice (DDB‐initiated mice). In the short‐term study, the animals were allocated into three groups: vehicle group (negative control), MNU group (positive control) and LGEO 500 mg kg^?1 (five times per week for 5 weeks) plus MNU group (test group). Blood samples were collected to analyze leukocyte DNA damage by comet assay 4 h after each MNU application at the end of weeks 3 and 5. The LGEO 500 mg kg^?1 treated group showed significantly lower (P < 0.01) leukocyte DNA damage than its respective positive group exposed to MNU alone at week 3. In the medium‐term study, DDB‐initiated mice were allocated into three groups: vehicle group (positive control) and LGEO 125 or 500 mg kg^?1 (five times per week for 6 weeks; test groups). At week 20, all animals were euthanized and mammary glands, colon and urinary bladder were processed for histopathological analyses for detection of preneoplastic and neoplastic lesions. A slight non‐significant effect of treatment with LGEO 500 mg kg^?1 in reducing development of alveolar and ductal mammary hyperplasia was found (P = 0.075). Our findings indicate that lemongrass essential oil provided protective action against MNU‐induced DNA damage and a potential anticarcinogenic activity against mammary carcinogenesis in DDB‐initiated female Balb/C mice. Copyright © 2010 John Wiley & Sons, Ltd.     

Chronic terbutaline treatment desensitizes β-adrenergic inhibition of lymphocyte activation in healthy volunteers

1 A substantial body of evidence has accumulated that β-adrenoceptor mediated increases in human lymphocyte cyclic AMP can inhibit activation of resting lymphocytes. The aim of this study was to determine whether this effect might desensitize during chronic β-adrenoceptor agonist treatment. We assessed the effects of 2 weeks treatment with the β₂-adrenoceptor agonist terbutaline (3 × 5 mg day^?1 p.o.) on isoprenaline-induced inhibition of concanavalin A-evoked lymphocyte activation in nine healthy male volunteers. Lymphocyte activation was determined by [³H]-thymidine incorporation (as a measure of proliferation), and inositol phosphate formation was assessed in [³H]-myo-inositol prelabelled lymphocytes in the presence of 10 mm LiCl. 2 Terbutaline treatment caused a significant reduction in isoprenaline (1 nm – 10 μm )-induced increases in lymphocyte cyclic AMP content; the maximal increase was 14 ± 3 pmol/10⁶ cells before and 7 ± 2 pmol/10⁶ cells (n= 9, P < 0.05) after terbutaline treatment. 3 The mitogen concanavalin A (Con A, 1–32 μg ml^?1)-induced increase in inositol phosphate formation was significantly enhanced after terbutaline treatment (max. increase before treatment: 255 ± 25% above basal; after treatment 453 ± 16% above basal; n= 9, P < 0.001), while isoprenaline (1 nm – 10 μm )-induced inhibition of Con A (16 μg ml^?1)-evoked increases in inositol phosphate formation was significantly reduced after the terbutaline treatment (max. inhibition before treatment: 22 ± 4%; after treatment 9 ± 1%, n= 9, P < 0.01). 4 Con A (1.25 – 10 μg ml^?1)-induced increases in [³H]-thymidine incorporation into the lymphocytes (as a measure of proliferation) was not affected by the terbutaline treatment. On the other hand, isoprenaline (1 nm – 1 μm )-induced inhibition of Con A (5 μg ml^?1)-evoked lymphocyte proliferation (max. inhibition: 33 ± 7%, n= 9) was almost completely abolished after the terbutaline treatment. 5 We conclude that chronic treatment with terbutaline desensitizes lymphocyte β₂-adrenoceptors and, therefore, the inhibitory effect of cyclic AMP on lymphocyte activation.     

Antigenotoxic activities of the natural dietary coumarins umbelliferone,herniarin and 7-isopentenyloxy coumarin on human lymphocytes exposed to oxidative stress

Abstract

The antigenotoxic effects of umbelliferone (UMB), herniarin (HER) and 7-isopentenyloxy coumarin (7-IP), common natural dietary coumarins, were evaluated on the human lymphocyte DNA damage using single-cell gel electrophoresis. H₂O₂-induced DNA break was measured based on the percentage of DNA in tail, and the antigenotoxic effects of the tested compounds were compared with that of ascorbic acid (10, 25, 50, 100 and 200?μM). UMB, HER and 7-IP did not show any genotoxicity, as compared to phosphate-buffered saline. Treatment with UMB, HER and 7-IP led to a significant reduction in the percentage of DNA in tail induced by H₂O₂ (p?<?0.001) at all concentrations. The presence of prenyl moiety in the chemical structure of 7-IP may contribute to its better antigenotoxic property, compared to UMB. The results of this study showed that 7-IP possessed the best antigenotoxic activity among the tested compounds.     

The binding of paracetamol to plasma proteins of man and pig

The binding of N-acetyl-4-aminophenol (paracetamol) to human and porcine plasma at both toxic and therapeutic concentrations was investigated by ultrafiltration and equilibrium dialysis over the range 50–300 μg ml^?1. Plasma protein binding occurred at paracetamol concentrations greater than 60 μg ml^?1. The extent of protein binding at a plasma concentration of 280 μg ml^?1 of the drug is between 15 and 21% for both pig and man. There is no appreciable binding to erythrocytes in either species over the whole concentration range studied.     

Effects of low‐dose exposure to pesticide mixture on physiological responses of the pacific oyster,Crassostrea gigas

This study investigated the effects on the physiology of Pacific oyster, Crassostrea gigas, of a mixture of pesticides containing 0.8 μg L^?1 alachlor, 0.6 μg L^?1 metolachlor, 0.7 μg L^?1 atrazine, 0.6 μg L^?1 terbuthylazine, 0.5 μg L^?1 diuron, 0.6 μg L^?1 fosetyl aluminum, 0.05 μg L^?1 carbaryl, and 0.7 μg L^?1 glyphosate for a total concentration of 4.55 μg L^?1. The total nominal concentration of pesticides mixture corresponds to the pesticide concentrations in the shellfish culture area of the Marennes‐Oleron basin. Two varieties of C. gigas were selected on the foreshore, based on their characteristics in terms of resistance to summer mortality, to assess the effects of the pesticide mixture after 7 days of exposure under controlled conditions. The early effects of the mixture were assessed using enzyme biomarkers of nitrogen metabolism (GS, glutamine synthetase), detoxification metabolism (GST, glutathione S‐transferase), and oxidative stress (CAT, catalase). Sublethal effects on hemocyte parameters (phagocytosis and esterase activity) and DNA damages (DNA adducts) were also measured. Changes in metabolic activities were characterized by increases in GS, GST, and CAT levels on the first day of exposure for the “resistant” oysters and after 3–7 days of exposure for the “susceptible” oysters. The formation of DNA adducts was detected after 7 days of exposure. The percentage of hemocyte esterase‐positive cells was reduced in the resistant oysters, as was the hemocyte phagocytic capacity in both oyster varieties after 7 days of exposure to the pesticide mixture. This study highlights the need to consider the low doses and the mixture of pesticides to evaluate the effects of these molecules on organisms. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 689–699, 2013.