In vitro effects of selected endocrine disruptors (DEHP, PCB118, BPA) on narrow-clawed crayfish (Astacus leptodactylus) primary cells.

Environmental pollutants with endocrine-disrupting effect are of global importance due to their contribution to the aethiologies of variety of complex diseases. These lipophilic pollutants are persistent in the environment and able to bioaccummulate in nontarget organisms. BPA, DEHP and PCB118 (dioxin-like PCB) are associated with endocrine disruption effects, while information on their effects on aquatic invertebrates are limited. In the current study, the effects of these compounds, which are ubiqutous and present at low concentrations in the environment, are studied in the primary hepatopancreas, muscle, gill, intestine and gonadal cultures of narrow-clawed crayfish (Astacus leptodactylus Eschscholtz, 1823), a widely distributed freshwater crayfish in Turkey with high economic importance. IC50 values following MTT assay ranged 0.27-12.61 nM; when compared with other tissues, the gonads were more affected with lower IC50 values. PCB118 induced higher cytotoxicity, while DEHP was the least toxic compound. This is the first study on the primary culture of A. leptodactylus¸ and the toxic effects of these compounds in this organism providing mechanistic insights on the responses and detoxification capacity of the organs. This study provides basis to unravel the mechanism of action of the tested EDCs in crayfish and improvement of cell culture conditions for ecotoxicity and screening assays.

Phthalate induced toxicity in prostate cancer cell lines and effects of alpha lipoic acid.

OBJECTIVE: The effects of dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di-n-butyl phthalate, benzylbutyl phthalate, di-2-ethylhexyl phthalate were investigated on human prostate cancer cell lines DU145 and PC3 in vitro. MATERIALS AND METHODS: Standards of dimethyl phthalate, diethyl phthalate, di-isobutyl phthalate, dibutyl phthalate, benzyl butyl phthalate, and di-ethyl hexyl phthalate were used. Alpha lipoic acid was used as antioxidant compound. DU145 and PC3 human prostate carcinoma cells were used. MTT assay were used for cytotoxicity assay. RESULTS: A low dose proliferative effect of phthalates in vitro was observed. With the hypothesis of the inhibition of aerobic glycolysis activity in cancer treatment, α-lipoic acid was applied to cells; where as a contrary to previous studies, no change in the cell proliferation was observed. In combination with ALA, at IC50 and lower doses, an increase of the cytotoxic effect was found for DIBP, DBP and BBP; while for DMP, DEP and DEHP, a decrease was observed for DU145 cells. In PC3 cells, a decrease was observed for DMP, DEP and DBPs; while no significant difference were observed for DEHP, DIBP and BBP. CONCLUSSION: The present study demonstrates preliminary information regarding the low dose proliferative effects of phthalates in prostate cancer in vitro (Tab. 2, Fig. 2, Ref. 65).

Effects of polychlorinated biphenyls 28, 30 and 118 on bovine spermatozoa in vitro.

Decline of semen quality due to endocrine-disrupting chemicals is of concern globally. Among endocrine disrupting chemicals (EDC), Polychlorinated biphenyls (PCB) are associated with reduced semen quality in various epidemiological studies. In this study, we evaluated the direct effects of selected PCBs (28, 30 and 118) on fresh spermatozoa of Simmetial bulls aged 2-4 years were evaluated in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and computer assisted semen analysis (CASA) (SCA; Microptics) analysis. IC50 values were found as 8.45, 5.45 and 9.55 ng/ml for PCB 28, 30 and 118, respectively. Total motility, progressive motility and viability decreased dependent on dose and duration of exposure (0, 2, 4 h). Total motility at IC50 doses decreased the most in PCB 28 (72.24%) followed by 30 (60.75%) and 118 (64.77%) at 2nd hour following exposure. Motility results were found to be in accordance with the vitality and morphology data where total abnormalities (especially reacted acrosome rate) were found to have increased.

Depletion of florfenicol and florfenicol amine residues in chicken eggs.

1. The aim of this study was to develop a suitable method for the analysis of florfenicol (FF) and its metabolite florfenicol amine (FFA) in chicken eggs and to determine FF and FFA residue depletion in eggs of laying hens. 2. The analytes were extracted from yolk, albumen and whole egg by phosphate buffer and ethyl acetate. Following purification, samples were analysed by high-performance liquid chromatography. 3. Fifty laying hens were divided into 5 groups, and each hen received doses of 20 mg/kg FF: Group 1 (received a single oral dose by gavage); Group 2 (a single intramuscular dose); Group 3 (a single subcutaneous dose); Group 4 (multiple oral doses for 3 d) and Group 5 (multiple oral doses for 5 d). 4. Limits of detection and of quantitation values were 1.94 and 6.45 g/10(9) g (ppb) for FF, respectively, and 0.48 and 1.58 ppb for FFA, respectively. Relative standard deviation values of intra-day and inter-day variation below 11% also confirmed the usefulness of the method for analysing FF and FFA in eggs. 5. From the first day of both oral and parenteral administration, FF and FFA were detected at 0.1% and 0.08% of dosage, respectively, and 57% of the drugs were eliminated from the egg yolk. Elimination time of FF was 8 d in Groups 1, 2 and 3; 9 d in Group 4 and 10 d in Group 5.

Determination of melamine in milk and dairy products by high performance liquid chromatography.

A simple, precise, accurate, and validated reverse-phase HPLC method was developed for the determination of melamine in milk (pasteurized and UHT milk) and dairy products (powdered infant formula, fruit yogurt, soft cheese, and milk powder). Following extraction with acetonitrile:water (50:50, vol/vol), samples were purified by filter (0.45 µm), separated on a Nucleosil C8 column (4.6 mm × 250 mm, 3 µm) with acetonitrile:10 mmol/L sodium L-octane sulfonate (pH 3.1; 15:85, vol/vol) as mobile phase at a flow rate of 1 mL/min, and determined by a photodiode array detector. A linear calibration curve was obtained in the concentration range from 0.05 to 5 mg/kg. Milk and dairy products were fortified with melamine at 4 levels producing average recovery yields of 95 to 109%. The limits of detection and quantification of melamine were 35 to 110 and 105 to 340 µg/kg, respectively. The method was then used to analyze 300 samples of milk and dairy products purchased from major retailers in Turkey. Melamine was not found in infant formulas and pasteurized UHT milk, whereas 2% of cheese, 8% of milk powder, and 44% of yogurt samples contained melamine at the 121, 694±146, and 294±98 µg/kg levels, respectively. These findings were below the limits set by the Codex Alimentarius Commission and European Union legislation. This is the first study to confirm the existence of melamine in milk and dairy products in Turkey. Consumption of foods containing these low levels of melamine does not constitute a health risk for consumers.

Detection of aflatoxin M1 concentrations in UHT milk consumed in Turkey markets by ELISA.

In this study, ELISA (Enzyme Linked Immunosorbent Assay) technique was used for detection of aflatoxin M1 in UHT milk sold in Bursa-TURKEY for consumption. A total of 50 samples of commercial UHT (Ultra High Temperature) whole milk were analyzed. Aflatoxin M1 residues were detected in all samples (100%) studied in different levels. The mean value was 101.2 +/- 53.8 ng L(-1). Although, 40 (80%) were below the limit, the remaining 10 (20%) were well above the limit permitted by European Community and Turkish Food Codex. Serious risks for public health exist from milk consumption. Therefore, milk has to be controlled periodically for AFM1 contamination. Also, dairy cow feeds should be stored in such a way that they do not become contaminated.

Study of the relationship between Androctonus crassicauda (Oliver, 1807; scorpiones, buthidae) venom toxicity and telson size, weight and storing condition

Scorpions are included in the order Scorpiones; class Arachnida. Lethal scorpions are mostly of the Buthidae family. Among these, species belonging to Androctonus, Leiurus and Mesobuthus genera cause most scorpion envenomations in Turkey. This study was performed aiming the production of antivenom by using Androctonus crassicauda telsons. Venom toxicity is related to telson weight, size, and storing condition (open or closed). Telsons of A. crassicauda were collected in Southeastern Anatolia (especially in Harran town, Sanliurfa), Turkey. They were separated according to weight, size, and storing condition - open (a) and closed (b). Venom solution was prepared by maceration of telsons. Swiss albino mice were used to determine the lethal dose 50% (LD50), which was as follows: Group 1a - 2.31mg; Group 1b - 2.66mg; Group 2a - 2.32mg; Group 2b - 2.66mg; Group 3a - 6.66mg; Group 3b - 6.88mg. Among the groups of telsons, the first and the second groups showed different characteristics. However, there were no differences between their toxicity. In the third group, a fourfold amount of telsons was used for toxicity. In other words, telsons weighting from 19.99 to 20mg (first group) and from 29.99 to 30mg (second group) presented similar LD50 values, and telsons weighting from 10 to 19.99mg (third group) showed a fourfold higher LD50 value. This difference was caused by the maturity of scorpions and venom toxicity was related to their size. The first and second groups were considered to be mature and the third group, not adult. Therefore, we can conclude that obtaining open telsons due to environmental factors was not effective for venom toxicity.

Optimization of antiscorpion venom production

The present study was carried out to produce highly efficient antivenom from a small number of telsons in a short time. Venom solution was prepared through maceration of telsons from Androctonus crassicauda (Olivier, 1807) collected in the Southeastern Anatolia Region, Turkey. Lethal dose 50% (LD50) of the venom solution injected into mice was 1 ml/kg (95% confidence interval; 0.8-1.3), according to probit analysis. Different adjuvants (Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, and 0.4% aluminium phosphate), at increasing doses and combined with venom, were subcutaneously injected into horses on days 0, 14, 21, 28, 35, and 42 of the experiment. Antivenom was collected from the immunized horses on days 45, 48, and 51 using the pepsin digestive method. The antivenom effective dose 50% (ED50) in mice was 0.5 ml (95% confidence interval; 0.40-0.58), according to probit analysis. It was concluded that 0.5 ml antivenom neutralized a venom dose 35-fold higher than the venom LD50. Thus, highly potent antivenom could be produced from about 238 telsons in 51 days.

Residues of gentamicin in eggs following medication of laying hens.

1. Gentamicin was injected subcutaneously and intramuscularly into 5 groups of 10 laying hens and its concentration was determined in albumen, yolk and whole egg. 2. Groups 1 and 3 were intramuscularly injected with doses of 10 and 25 mg/kg while groups 2, 4 and 5 were subcutaneously injected with doses of 10, 25 and 50 mg/kg, respectively. 3. The final gentamicin concentration in albumen was measured on d 3 for groups 1 and 2; on d 4 for groups 3 and 4, and on d 5 for group 5. Concentrations in yolk and whole egg were measured on d 7, 10 and 12. 4. Gentamicin recovery was as follows: 2% in groups 1 and 2, 2.5% in groups 3 and 4, and 3% in group 5. 5. Most of the residue (approximately 90%) was recovered from the yolk.