Bioactivity of two major constituents isolated from the essential oil of Artemisia judaica L

The essential oil of Artemisia judaica L., grown on Sinai Peninsula of Egypt, was extracted via hydrodistillation. Chromatographic separation on repeated silica gel columns led to isolate two compounds namely piperitone and trans-ethyl cinnamate. Insecticidal, antifeedant and antifungal properties of the isolated compounds were examined. Both compounds showed pronounced insecticidal and antifeedant activity against the third instar larvae of Spodoptera littoralis (Boisd). trans-Ethyl cinnamate (LD₅₀ = 0.37 μg/larva) was more toxic than piperitone (LD₅₀ = 0.68 μg/larva). The two isolated compounds revealed antifeedant activity in a concentration dependent manner, with complete feeding inhibition at a concentration of 1000 μg/ml. When tested for antifungal activity against four plant pathogenic fungi, the isolated compounds exhibited a moderate to high activity.     

Arachidonate lipoxygenase inhibitors in guinea-pig isolated trachea. Effects on contractions to antigen and various agonists

We compared the effects of the leukotriene (LT) D₄ receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase inhibitor indomethacin (5 μM), and by arachidonic acid (AA), melittin and LTD₄. FPL55712 (0.1 and 1 μM) inhibited contractions induced by AA (100 μM) and the phospholipase A₂ activator melittin (3 μg/ml), while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 μM) was a more effective inhibitor of the melittin response than the response. FPL55712 inhibited contractions induced by OA (100 μg/ml) more than by A23187 (1 μg/ml), and these inhibitory effects of FPL55712 were much less in the presence of l-serine-borate complex (45 mM), an inhibitor of LTC₄ conversion to LTD₄. NDGA (10 μM) had no significant effect on the OA response, whereas the lipoxygenase inhibitors 1-phenyl-3-pyrazolidone (phenidone, 10 μM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 μM) clearly inhibited it. In contrast, NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA, had a large inhibitory effect on LTD₄-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig trachea inhibitors of LTD₄-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.     

Simultaneous determination of zidovudine and its monophosphate in mouse plasma and peripheral red blood cells by high-performance liquid chromatography

Novel prodrugs for the intracellular delivery of zidovudine monophosphate (AZTMP) have recently been designed. To investigate the bioconversion and pharmacokinetic profiles of these compounds, an analytical method for the simultaneous determination of zidovudine (AZT) and AZTMP in mouse plasma and peripheral red blood cells was developed. Mouse whole blood samples were treated with TBAHS, EDTA and NaH₂PO₄, and separated into plasma and red blood cell portions. Samples were processed by solid-phase extraction using Bond Elut C₁₈ cartridges. Chromatography was performed using an Hypersil ODS column and a mobile phase of 2.9% (v/v) acetonitrile and 97.1% (v/v) phosphate buffer, pH 7.50, with UV detection at 267 nm. The average extraction recoveries of AZTMP and AZT in plasma were approximately 85% and 97% over their linear ranges of 0.05–5 μg/ml and 0.125–25 μg/ml, respectively. Extraction recoveries of AZTMP and AZT from peripheral red blood cells averaged 56 and 69% over their linear ranges of 0.125–5 μg/ml and 0.125–25 μg/ml, respectively. The accuracy of the assay was 90–100%. The intra- and inter-day variations of the assay were less than 14%. The analytical method was found to be applicable, reliable and suitable for pharmacokinetic studies.     

Effects of genistein and lavendustin on reproductive processes in domestic animals in vitro

The aim of our experiments was to study the influence of genistein [tyrosine kinase (TK) inhibitor with estrogenic activity] and lavendustin A (TK inhibitor without estrogenic activity) on female reproductive processes in domestic animals in vitro. It was found that genistein (0.001–1 μg/ml) increased IGF-I release by cultured bovine and porcine granulosa cells, but decreased its secretion by rabbit granulosa cells (0.01–10 μg/ml). Genistein stimulated progesterone secretion by bovine and rabbit granulosa cells (at 0.01–10 μg/ml), estradiol output by rabbit granulosa cells (at 1 μg/ml) and porcine ovarian follicles (at 10 μg/ml), as well as cAMP production by bovine (at 0.001–1 μg/ml) and rabbit (at 1 μg/ml) granulosa cells. No effects of genistein (at 10 μg/ml) on PGF-2 alpha and progesterone release by porcine ovarian follicles were observed. Genistein significantly (P < 0.05) stimulated the reinitiation and completion of nuclear maturation of porcine oocytes (at 5 μg/ml), as well as the preimplantation development of rabbit zygotes (at 1 μg/ml). Lavendustin A (0.001–1 μg/ml) increased IGF-I release by bovine (but not by porcine) granulosa cells, cAMP release by bovine granulosa cells, and PGF-2 alpha output by porcine ovarian follicles (at 10 μg/ml). Lavendustin (at 1 μg/ml) had no significant effect on IGF-I release by porcine granulosa cells, on estradiol and cAMP output by rabbit granulosa cells, or on progesterone secretion by porcine follicles (at 10 μg/ml). Inhibitory actions of lavendustin (at 10 μg/ml) on estradiol secretion by porcine follicles were also found. Furthermore, lavendustin, like genistein, promoted the reinitiation and completion of meiosis in porcine oocytes. The present study demonstrates a predominantly stimulatory effect of TK inhibition on endocrine and generative processes in domestic animals. The majority of these effects are similar for both compounds, indirectly suggesting that their action is due to tyrosine kinase inhibition and protein kinase A-stimulation, rather than estrogenic activity.     

Effects of cordycepin on macromolecular synthesis and development in the preimplantation mouse embryo

Investigations were conducted to test the effects of cordycepin, a naturally-occurring analog of adenosine, on gene activity in preimplantation mouse embryos. Embryos were explanted into culture at the 2-cell, morula and blastocyst stages, and incubated in the absence or presence of cordycepin (5–100 μg/ml) to determine the effects of the drug on continued development and macromolecular synthesis. Cordycepin at concentrations exceeding 10 μg/ml caused a dose-responsive inhibition of cleavage and blastulation of embryos in culture. Exposure of morulae and blastocysts to cordycepin concentrations of 10–100 μg/ml produced a dose- and time-dependent suppression of RNA synthesis as measured by incorporation of [³H]uridine. Suppression in blastocyst-stage embryos was enhanced by preincubation, and reached 70% after 4 h at 100 μg/ml. Cordycepin (50–100 μg/ml) reduced synthesis of major RNA components detected by electrophoresis, blocked incorporation of radioactivity into fractions bound by olido(dT)-cellulose, and produced a time- and dose-dependent reduction of protein synthesis in blastocysts, causing a maximum inhibition of 25% after 4 h of preincubation at 50 μg/ml.     

Anticancer activities of a chemically sulfated polysaccharide obtained from Grifola frondosa and its combination with 5-Fluorouracil against human gastric carcinoma cells

Chemically sulfated polysaccharide (S-GAP-P) was derived from water-insoluble polysaccharide of Grifola frondosa mycelia. In this research, we investigated the anticancer effects of S-GAP-P and its combination with 5-fluorouracil (5-FU) on human gastric carcinoma SGC-7901 cells. Results showed that S-GAP-P distinctly inhibited SGC-7901 cells growth in a dose-dependent manner and induced cell apoptosis evidenced by characteristic DNA ladder and sub-G₀/G₁ peak. Furthermore, the combination of S-GAP-P (10–50 μg/ml) with 1 μg/ml 5-FU resulted in a significant inhibition on SGC-7901 cells growth, meaning the beneficial interaction between the two drugs. All these results suggested that S-GAP-P has evident anticancer activity through apoptotic induction and could significantly accelerate the anticancer activity of 5-FU.     

Effects of cytochalasin B on cell division and vacuole formation in Tetrahymena pyriformis GL

Cytochalasin-B (Cyt-B) was tested for its effect on cell division and vacuole formation in Tetrahymena. Little effect was found on cell division in the synchronous cell system in concentrations up to 37 μg/ml; however, slight delay was caused by 71 μg/ml. As measured by particle uptake, much lower concentrations, 7–8 μg/ml, caused significant inhibition of vacuole formation in exponentially multiplying and in starved cells, 16.6 or 37 μg/ml caused strong inhibition. This effect was immediate and completely reversible. The presence of Cyt-B caused starvation of Tetrahymena. The essential absence of inhibition of cell division by Cyt-B may reflect that the drug can enter the cell only by way of vacuoles.     

Effect of flavan-3-ols on in vitro egg hatching, larval development and viability of infective larvae of Trichostrongylus colubriformis

The effects of flavan-3-ols (the monomer units of condensed tannins (CT)) and their galloyl derivatives on the viability of eggs, the development of first stage (L1) larvae, and the viability of the infective larvae of Trichostrongylus colubriformis were investigated under in vitro conditions. Each of the flavan-3-ol gallates showed some inhibition of egg hatching at 100 μg/ml, and 100% inhibition at 1000 μg/ml, with epigallocatechin gallate being the most effective in the egg hatch (EH) assay. In contrast, none of the flavan-3-ols were able to completely inhibit egg hatching. The flavan-3-ols and galloyl derivatives dose-dependently inhibited the development of infective larvae as assessed by the larval development (LD) assay. A larval migration inhibition (LMI) assay was used to assess the effect of flavan-3-ols and their galloyl derivatives on the motility of the infective third-stage (L3) larvae of T. colubriformis. In general, the flavan-3-ol gallates were more effective than the flavan-3-ols at immobilising the infective larvae as evidenced by their ability to inhibit more (P<0.05–0.01) larvae from passing through the LMI sieves. At 500 μg/ml, epigallocatechin gallate inhibited significantly more (P<0.1) larvae from passing through the sieves than did catechin gallate, epicatechin gallate, or gallocatechin gallate. Comparisons were made between the flavan-3-ols and their galloyl derivatives with the in vitro effects of CT extracts from several forage legumes, which have exhibited effects on parasites in vivo. The forage legumes tested at 200–500 μg/ml reduced the proportion of eggs that hatch, with comparable results to those obtained using the flavan-3-ols. The activities may be influenced by the prodelphinidin: procyanidin (PD:PC) ratios: CT extracts from Lotus pendunculatus and sainfoin have PD:PC ratios of 70:30 and 77:23, respectively, whereas the less active CT extract from Lotus corniculatus has a PD:PC ratio of 27:73. The active CT extracts from forage legumes have epigallocatechin as the dominant flavan-3-ol extender unit, and epigallocatechin is the most active flavan-3-ol in both the EH and LD assays.     

The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays

Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l⁻¹). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml⁻¹ LDH and 0.8 U ml⁻¹ LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC₁₀ of 800 nM (213 μg l⁻¹) and a minimum inhibition detectable (MID) limit of 650 nM (173 μg l⁻¹). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml⁻¹ LDH, 0.8 U ml⁻¹ LOD and 0.1 U ml⁻¹ GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC₁₀ limit was 150 nM (39.9 μg l⁻¹) and the MID was 100 nM (26.6 μg l⁻¹). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.     

Metal complexes of crosslinked chitosans Part II. An investigation of their hydrolysis to chitooligosaccharides using chitosanase

This paper investigates the behavior of crosslinked chitosans and metal-complexed crosslinked chitosans under similar hydrolytic conditions. Crosslinked chitosans with trimellitic anhydride, diisocyanatohexane, and dibromodecane as crosslinking agents under heterogenous reaction conditions were used as metal complexing agents by equilibrating them with metal salts such as ZnCl₂, MnSO₄, CuSO₄, CdSO₄, Pb(NO₃)₂, and HgCl₂. Crosslinked chitosan without metal complexation had the same hydrolytic behavior as uncrosslinked chitosan. However, when the crosslinked chitosans were complexed with metals, their rates of hydrolysis and extent of hydrolysis were significantly reduced. Thus, while for chitosan about 840 μg/ml reducing sugar was produced in 4 h time, and 780 μg/ml was produced for diisocyanatohexane crosslinked chitosan, only 400 μg/ml and 320 μg/ml reducing sugars were produced for cadmium sulfate with crosslinked chitosan and diisocyanatohexane crosslinked chitosan, respectively. Similar results are obtained for other crosslinking agents. Studies on preincubation of the metal with the enzyme show that of the metals studied, Mn has no effect on preincubatioin with the enzyme, Hg, Cd, Pb, and Cu completely deactivates the enzyme, while Zn reduces the enzyme activity by about 43.3%. Preincubation of the metal salts with the chitosan shows that Hg and Cu completely deactivate the molecule from enzyme hydrolysis, Cd and Zn inactivate it to the extent of 56.8% and 43.3%, respectively, while Mn has no effect. Availability of the amino functions seems to be a key feature for the chitosanase to hydrolyze the chitosan polymer. This was also proved by the significant increase in the extent of hydrolysis for chitosan samples with 88% (final value 1120 μg/ml reducing sugar) and 85% deacetylation (final value 840 μg/ml reducing sugar). HPIC studies of the products show that a variety of oligomers are produced in the chitosanase enzyme hydrolytic reaction.     

Tradescantia stamen hair mutation bioassay

The Tradescantia stamen hair mutation (Trad-SH) assay (clone 4430) was evaluated for its efficiency and reliability as a screen for mutagens in an IPCS collaborative study on plant systems. Four coded chemicals, i.e. azidoglycerol (AG, 3-azido-1,2-propanediol), N-methyl-N-nitrosourea (MNU), sodium azide (NaN₃) and maleic hydrazide (MH) were distributed by the Radian Corporation to the five laboratories in five different countries for testing mutagenicity. Pink mutations were scored between the 7th and 14th day according to a standard protocol. Test results from the five individual laboratories were analyzed and compared after decoding. One out of the two laboratories that conducted tests on AG demonstrated that AG is a mutagen with genetically effective doses ranging from 50 to 100 μg/ml. MH yielded positive responses in all laboratories but no linear dose-response pattern was observed. The effective dose range for MH was between 1 and 45 μg/ml. The mutagenicity of MNU was reported by five laboratories in the dose range between 10 and 80 μg/ml. NaN₃, which exhibited a relatively high degree of toxicity, elicited a positive mutagenic response in three of the five laboratories in which it was tested. As with MNU the effective dose for NaN₃ ranged between 3 and 80 μg/ml. The results from the current study substantiate the Trad-SH assay as a reliable system for screening chemicals for their potential mutagenic effects. Although the study was carried out exclusively under laboratory conditions, a survey of the current literature would indicate that the Trad-SH assay could be an effective in situ monitor of gaseous, liquid, and radioactive pollutants as well.     

Melatonin reduces both basal and bacterial lipopolysaccharide-induced lipid peroxidation in vitro

The protective effect of melatonin against lipopolysaccharide (LPS)-induced oxidative damage was examined in vitro. Lung, liver, and brain malonaldehyde (MDA) plus 4-hydroxyalkenals (4-HDA) concentrations were measured as indices of induced membrane peroxidative damage. Homogenates of brain, lung, and liver were incubated with LPS at concentrations of either 1, 10, 50, 200, or 400μg/ml for 1 h and, in another study, LPS at a concentration of 400 μg/ml for either 0, 15, 30, or 60 min. Melatonin at increasing concentrations from 0.01–3 mM either alone or together with LPS (400μg/ml) was used. Liver, brain, and lung MDA + 4-HDA levels increased after LPS at concentrations of 10, 50, 200 or 400 μg/ml; this effect was concentration-dependent. The highest levels of lipid peroxidation products were observed after tissues were incubated with an LPS concentration of 400 μg/ml for 60 min; in liver and lung this effect was totally suppressed by melatonin and partially suppressed in brain in a concentration-dependent manner. In addition, melatonin alone was effective in brain at concentrations of 0.1 to 3 mM, in lung at 2 to 3 mM, and in liver at 0.1 to 3 mM; in all cases, the inhibitory effects of melatonin on lipid peroxidation were always directly correlated with the concentration of melatonin in the medium. The results show that the direct effect of LPS on the lipid peroxidation following endotoxin exposure is markedly reduced by melatonin.     

In vitro antiplasmodial activity of extract and constituents from Esenbeckia febrifuga, a plant traditionally used to treat malaria in the Brazilian Amazon

Esenbeckia febrifuga (Rutaceae) is a plant traditionally used to treat malaria in the Brazilian Amazon region. Ethanol extract of stems displayed a good antiplasmodial activity against Plasmodium falciparum strains W-2 (IC₅₀ 15.5±0.71 μg/ml) and 3 D7 (IC₅₀ 21.0±1.4 μg/ml). Two coumarins (bergaptene 1 and isopimpinellin 2), five alkaloids (flindersiamine 3, kokusaginine 4, skimmiamine 5, γ-fagarine 6 and 1-hydroxy-3-methoxy-N-methylacridone, 7), besides a limonoid (rutaevine 8), have been isolated for the first time from this species. Antiplasmodial activity of compounds 3, 5–8 has been evaluated in vitro against P. falciparum strains (W-2 and 3D7) and the furoquinolines 5 and 6 were the most potent displaying IC₅₀ values <50 μg/ml; flindersiamine (3) showed a weak activity while alkaloid 7 and rutaevine (8) were inactive (IC₅₀>100 μg/ml).     

Effects of tannic acid and its related compounds on food mutagens or hydrogen peroxide-induced DNA strands breaks in human lymphocytes

The effect of tannic acid (TA), gallic acid (GA), propyl gallate (PA) and ellagic acid (EA) on DNA damage in human lymphocytes induced by food mutagens [3-amino-1-methyl-5H-pyrido (4,3-b) indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimadazo (4,5-b) pyridine (PhIP) or H₂O₂ was evaluated by using single-cell electrophoresis (comet assay). The toxicity of these tested compounds (0.1–100 μg/ml) on lymphocytes was not found. These compounds did not cause DNA strand breaks at lower concentrations of 0.1–10 μg/ml. At a concentration of 100 μg/ml, TA and GA exhibited slight DNA damage, whereas PA and EA showed no DNA strand breaks. TA and its related compounds decreased the DNA strand breaks induced by Trp-P-2, PhIP or H₂O₂ at concentrations of 0.1–10 μg/ml. DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycoslase (FPG)] were used to examine the levels of oxidised pyrimidines and purines in human lymphocytes induced by H₂O₂. All the compounds at 10 μg/ml can reduce the level of FPG sensitive sites. However, only EA inhibited the formation of EndoIII sensitive sites. The results indicated that these compounds can enhance lymphocytes resistance towards DNA strand breaks induced by food mutagens or H₂O₂ in vitro.     

Relationship between cellular RecA protein concentration and untargeted mutagenesis in Escherichia coli

We measured the production of untargeted mutations in the cI and cII genes of untreated λ phage undergoing a lytic cycle in UV-irradiated bacterial hosts. As previously shown, treatment with 4 μg/ml of rifampicin during post-irradiation incubation inhibited amplification of the RecA protein in these cells. In addition, we observed a decreased mutation rate compared to the untreated, irradiated bacteria. Treatment with 4 μg/ml or 8 μg/ml rifampicin did not prevent the UV induction of the umuDC operon, as judged by assay of β-galactosidase activity in a umuC-lacZ fusion strain. In contrast, the UV-induction of β-galactosidase in the sulA-lacZ fusion strain was decreased by 4 μg/ml rifampicin. The inhibition of untargeted mutagenesis by this drug treatment was also observed in a strain constitutive for SOS functions (lexA (Def)) as well as ina RecA-overproducing plasmid strain, that blocks induction of heat-shock proteins, factor(s) in wild-type recA⁺ cells. An htpR165-carrying strain, that blocks induction of heat-shock proteins, exhibited normal UV-promoted mutagenesis. A correlation was observed between the cellular concentration of RecA protein, increased spontaneously by a temperature shift in a lexA(Ts) strain, and the extent of UV-promoted untargeted mutagenesis. These results suggest a mechanistic role of RecA protein in this process.     

Involvement of steroid hormones in bovine oocytes maturation in vitro.

Influences of steroid hormone additions or of their binding by specific antisera on nuclear maturation and subsequent fertilization and cleavage of bovine oocytes were studied in vitro. It was found that progesterone in doses of 50 ng/ml, 250 ng/ml, 1 μg/ml or 5 μg/ml stimulates reinitiation and in doses of 1 or 5 μg/ml stimulates further development of meiosis. Antiserum to progesterone had opposite effects on nuclear maturation, but has no influence on the ability of matured oocytes to subsequent fertilization and cleavage. Testosterone additions (10 ng, 100 ng, 1 μg or 5 μg/ml) did not influence nuclear maturation, but antiserum to this hormone inhibited both meiosis reinitiation and completion, as well as lowered the rate of oocytes fertilized and embryos obtained. Estradiol (5, 50, 100 or 500 ng or 5 μg/ml) treatment stimulated reinitiation, but not nuclear maturation. Antiserum to estradiol activated both reinitiation, development and completion of meiosis, but the cells matured by estradiol deficit were as a rule uncapable of fertilization and further cleavage. Estradiol addition (1 μg/ml) to maturation medium together with FSH (10 μg/ml) (but not of FSH alone) lead to a significantly higher rate of fertilization and cleavage of matured cells.

Results obtained suggest (1) relative independence of reinitiation, further development of nuclear maturation and cytoplasmic maturation regulation in bovine oocytes as well as (2) the involvement of steroid hormones in these three processes.     

The impact of long-term past exposure to elemental mercury on antioxidative capacity and lipid peroxidation in mercury miners

Limited information is available on the effects of chronic mercury exposure in relation to the risk of cardiovascular disease (CVD). It is known from in vitro and in vivo studies that Hg can promote lipid peroxidation through promotion of free radical generation, and interaction with antioxidative enzymes and reduction of bioavailable selenium. The objective of the study was to test the hypothesis that long-term past occupational exposure to elemental Hg (Hg⁰) can modify antioxidative capacity and promote lipid peroxidation in miners.

The study population comprised 54 mercury miners and 58 workers as the control group. The miners were examined in the post-exposure period. We evaluated their previous exposure to Hg⁰, the putative appearance of certain nonspecific symptoms and signs of micromercurialism, as well as the main behavioural and biological risk factors for CVD, and determined: 1) Hg and Se levels in blood and urine, 2) antioxidative enzymes, Cu/Zn superoxide dismutase (CuZn-SOD), catalase (CAT), and selenoenzyme glutathione peroxidase (GSH-Px) activity in erythrocytes as indirect indices of free radical activity, 3) pineal hormone melationin (MEL) in blood and urine, and 4) lipid hydroperoxides (LOOHs) and malondialdehyde (MDA) as lipid peroxidation products.

The mercury miners were intermittently exposed to Hg⁰ for periods of 7 to 31 years. The total number of exposure periods varied from 13 to 119. The cumulative U-Hg peak level varied from 794-11,365 μg/L. The current blood and urine Hg concentrations were practically on the same level in miners and controls. Miners showed some neurotoxic and nephrotoxic sequels of micromercurialism. No significant differences in behavioural and biological risk factors for CVD were found between miners and controls. A weak correlation (r = 0.36, p < 0.01) between systolic blood pressure and average past exposure U-Hg level was found. The mean P-Se in miners (71.4 μg/L) was significantly lower (p < 0.05) than in the controls (77.3 μg/L), while the mean U-Se tended to be higher (p < 0.05) in miners (16.5 μg/g creatinine) than in the controls (14.0 μg/g creatinine). Among antioxidative enzyme activities, only CAT in erythrocytes was significantly higher (p < 0.01) in miners (3.14 MU/g Hb) than in the controls (2.65 MU/g Hb). The mean concentration of B-MEL in miners (44.3 ng/L) was significantly higher (p < 0.01) than in the controls (14.9 ng/L). The mean value of U-MEL sulphate (31.8 μg/L) in miners was significantly lower (p < 0.01) than in the control group (46.9 μg/L). Among the observed lipid peroxidative products, the mean concentration of U-MDA was statistically higher (p < 0.01) in miners (0.21 μmol/mmol creatinine) than in the controls (0.17 μmol/mmol creatinine).

In the group of miners with high mercury accumulation and the presence of some nonspecific symptoms and signs of micromercurialism, the results of our study partly support the assumption that long-term occupational exposure to Hg⁰ enhances the formation of free radicals even several years after termination of occupational exposure. Therefore, long-term occupational exposure to Hg⁰ could be one of the risk factors for increased lipid peroxidation and increased mortality due to ischaemic heart disease (ICH) found among the mercury miners of the Idrija Mine.     

In vitro antiprotozoal activity of the lipophilic extracts of different parts of Turkish Pistacia vera L.

Thirteen lipophilic extracts prepared with n-hexane from various parts of Pistacia vera L. tree (Anacardiaceae) growing in Turkey were screened for their in vitro activity against four parasitic protozoa, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum. Melarsoprol, benznidazole, miltefosine, artemisinin and chloroquine were used as reference drugs. The cytotoxic potentials of the extracts on rat skeletal myoblast (L6) cells were also assessed and compared to that of podophyllotoxin. The screening method employed was medium-throughput, where the extracts were tested at two concentrations, at 0.8 and 4.8 μg/ml (T. brucei rhodesiense, L. donovani and Plasmodium falciparum), or at 1.6 and 9.7 μg/ml (T. cruzi and L6 cells). At 4.8 μg/ml concentration, the branch extract of Pistacia vera (PV-BR) significantly inhibited (77.3%) the growth of L. donovani, whereas the dry leaf extract (PV-DL) was active against Plasmodium falciparum (60.6% inhibition). The IC₅₀ values of these extracts were determined as 2.3 μg/ml (PV-BR, L. donovani) and 3.65 μg/ml (PV-DL, Plasmodium falciparum). None of the extracts possessed cytotoxicity on mammalian cells.     

Bromodeoxyuridine (BrdU) template and thymodine pool effects on high frequencies of sister-chromatid exchange (SCE) in Bloom syndrome cells and a mutant cell line (AsHa) originated from ataxia telangiectasia

The effect of bromodeoxyuridine (BrdU)-substituted DNA template and thymidine (dT) pool on excess sister-chromatid exchanges (SCEs) was studied in Bloom syndrome (BS) cells and an ataxia telangiectasia (AT)-derived mutant cell line (AsHa). When BS endomitotic cells were labeled with low and high (or high and low) BrdU concentrations during S₁ and S₂, only the BrdU concentration during S₁ phase affected the observed SCE. In BS cells about a 10-fold increase in SCEs occurs during or following replication on a BrdU-substituted template (high-high and high-low BrdU labeling) relative to the normal DNA template. SCEs decreased to about half in AsHa cells labeled with various BrdU doses (40, 60, 80 and 100 μg/ml) during only S₁, compared with those labeled during S₁ and S₂. Co-cultivation of AsHa and BS cells resulted in a significant reduction in SCE level from 70 to 13–17 in BS cells, lowered the BrdU concentrations necessary for sister-chromatid differential (SCD) staining from 40 to 10 μg/ml with normal SCE level and resulted in decreased level of SCEs at high BrdU concentrations (80–100 μg/ml) 12–14 SCE) in AsHa cells, compared with the originally increased SCE level (36.65 SCE at 100 μg/ml) without co-culture. However, co-cultivation between AsHa and normal cells lowered the BrdU dose necessary for SCD staining from 40 to 30 μg/ml; the dT pool possibly balanced at this level, which is clearly higher than that at co-cultivation between AsHa and BS cells. The reason for the very high BrdU doses needed to achieve SCD would seem to be that AsHa cells have high levels of thymidylate (TMP) synthetase, which maintain a large endogenous thymidine pool. This has been confirmed by direct measurement. These findings strongly support that excess and decreased dT pools are closely related to the condition necessary for high SCE induction.