Lucie Bláhová

Enantioselective effects of alpha-hexachlorocyclohexane (HCH) isomers on androgen receptor activity in vitro

Alpha-hexachlorocyclohexane (alpha-HCH), a part of the HCH pesticide mixture, is one of the most widespread persistent organic pollutants. Interestingly, only limited number of studies addressed the toxicity of alpha-HCH and the effects of its individual optical isomers have not been investigated in detail. In the present study we separated two alpha-HCH enantiomers by preparative HPLC and studied their activities towards androgen receptor (AR) using the MDA-kb2 cell line stably transfected with the luciferase reporter gene under the control of AR. There was no direct effect of alpha-HCH on AR but both isomers significantly suppressed the activity of AR in co-exposure with the natural ligand dihydrotestosterone in a concentration-dependent manner. One of the enantiomers appeared to be more active at lower concentration, which was also supported by the molecular modeling calculations with AR that showed a slight difference in estimated free energy of binding and inhibition constant between two enantiomers. Although studies with other pesticides demonstrated strong enantioselective differences in toxicity, the present research shows rather minor differences in modulations of AR by both alpha-HCH enantiomers. For the first time, enantioselective effects of alpha-HCH were demonstrated and the results suggest interaction with multiple regulatory events controlling the AR activity. Full elucidation of the toxicity mechanism will require further research.

Degradation of natural toxins by phthalocyanines–example of cyanobacterial toxin, microcystin

Phthalocyanines (Pcs) are promising photosensitizers for use in various branches of science and industry. In the presence of visible light and diatomic oxygen, phthalocyanines can react to produce singlet oxygen, a member of reactive oxygen species able to damage different molecules and tissues. The aim of this study was to investigate the ability of phthalocyanines to degrade natural toxins in the presence of visible light. As the representative of hardly degradable toxins, a group of cyanobacterial peptide toxins--microcystin-LR--was chosen for this study. According to our results, phthalocyanines are able to degrade 61.5% of microcystins within a 48-hour incubation (38% of microcystins was degraded after 24 h and 24% after 12 h of incubation). Although other oxidants like hydrogen peroxide or ozone are able to degrade microcystins within several hours, we assume that by optimizing the spectrum emitted by light source and by changing the absorption characteristics of Pcs, microcystins degradation by phthalocyanines could be more effective in the near future.

Toxicity of clomazone and its formulations to zebrafish embryos (Danio rerio)

Herbicides are the most widely used group of pesticides but after reaching water bodies they are able to cause adverse effects on non-target organisms. Different formulations using the same active ingredient are frequently available, which raises the issue of potential influence of different formulation types on herbicide toxicity. The present study evaluated the toxicity and teratogenic effects of the active ingredient clomazone and its two formulations (Rampa® EC and GAT Cenit 36 CS, both containing 360g a.i./l of clomazone) on zebrafish embryos. The crucial difference between the two formulation types is the way of active substance release. This investigation is the first report on zebrafish embryotoxicity of both clomazone and its formulations. The technical active ingredient and formulations caused mortality and diverse teratogenic effects, showing different levels of toxicity. The LC50 values for the technical ingredient, Rampa® EC and GAT Cenit 36 CS were 61.4, 9.6 and 92.5mg a.i./l, respectively. Spontaneous movements in 22 hpf embryos decreased under exposure to both the technical ingredient and formulations. A significant number of underdeveloped embryos was detected after exposure to clomazone and Rampa® EC, while no underdevelopment was noted in embryos exposed to GAT Cenit 36 CS. Exposure to the technical ingredient and formulations led also to a series of morphological changes and interfered with the growth of zebrafish embryos. The EC50 based on detection of edemas, spine and tail tip deformations and gas bladder absence (120hpf) was 12.1, 10.1 and 24.1mg/l for technical clomazone, Rampa® EC and GAT Cenit 36 CS, while teratogenicity index (TI) based on LC50/EC50 ratio was 5.1, 1 and 3.8, respectively. The data in this study showed that the emulsifiable concentrate formulation (Rampa® EC) caused statistically significantly higher toxicity, and the aqueous capsule suspension (GAT Cenit 36 CS) lower toxicity than technical clomazone. It indicates that different formulations with the same active ingredient may have different environmental impacts, which is why risk assessment based only on active ingredient toxicity might not be sufficient in terms of preventing formulation effects on the environment.

Phytoestrogens in milk: Overestimations caused by contamination of the hydrolytic enzyme used during sample extraction.

Isoflavones are natural phytoestrogens with antioxidant and endocrine-disrupting potencies. Monitoring of their levels is important to ensure the high quality and safety of food, milk, and dairy products. The efficiency and accuracy of phytoestrogen analyses in complex matrices such as milk depend on the extraction procedure, which often uses hydrolysis by means of the β-glucuronidase/sulfatase enzyme originating from Helix pomatia. The present study reveals that the commercially available hydrolytic enzyme is contaminated by several phytoestrogen isoflavones (genistein, daidzein, formononetin, and biochanin A) and their metabolite equol, as well as flavones (naringenin and apigenin) and coumestrol. We show that the concentrations of daidzein and genistein in the enzyme could have impaired the results of analyses of the main isoflavones in several previously published studies. Of 8 analyzed compounds, only equol was confirmed in the present study and it serves as a reliable marker of phytoestrogens originating from cow feed. Critical reassessment of phytoestrogen concentrations in milk is needed because several previously published studies might have overestimated the concentrations depending on the extraction procedure used.

Assessment of non-derivatized β-N-methylamino-l-alanine (BMAA) neurotoxin in free form in urine of patients with nonspecific neurological symptoms

Abstract The beta‐N‐methylamino‐l‐alanine (BMAA) is a non‐proteinogenic amino acid discussed to be produced by cyanobacteria forming harmful blooms. Since BMAA is suspected etiological agent in neurodegenerative diseases, there is a need to study and validate whether and in what concentrations can BMAA be present in human tissues. The aim of the present study was to validate analytical and extraction procedures for quantification of non‐derivatized BMAA in the urine using liquid chromatography and commercial ELISA Kit. The study was focused on BMAA in different forms ‐ dissolved, protein associated and total. The validated protocol included SPE followed by HILIC MS/MS for analyses of non‐derivatized free form of BMAA with a limit of quantification 20 ng/mL. The methods for other BMAA forms (i.e. protein‐associated and total) were also assessed but high matrix interferences did not allow their implementation. The method was used for analyses of free BMAA in 23 urine samples from healthy volunteers and psychiatric patients suffering from nonspecific neurological symptoms. Traces of BMAA were suspectedly detected in a single urine sample but they were not unequivocally proved according to all conservative analytical criteria. BMAA was also not confirmed in a repeatedly collected sample from the same person. The evaluated commercial BMAA ELISA Kit (Abraxis) was not suitable for determination of BMAA in extracted urine samples because of systematically highly false positive results. In agreement with recent findings, analyses of BMAA appear to methodologically challenging, and further research on BMAA in human tissues (or its precursors with potency to form BMAA under natural conditions or ‐ eventually ‐ during sample processing) is needed to clarify its potential ethiological role in neurodegenerative diseases. HighlightsMethod for analysis of free non‐derivatized BMAA in human urine based on SPE and HILIC LC MS/MS is presented.Protein‐associated and total BMAA forms could not be assessed due to matrix interferences.BMAA was suspected but not unequivocally proved in a single sample of a patient with neurological symptoms.Commercial ELISA Kit was not suitable for BMAA because of highly false positive results.

Impact of acute and subchronic inhalation exposure to PbO nanoparticles on mice

Abstract Lead nanoparticles (NPs) are released into air from metal processing, road transport or combustion processes. Inhalation exposure is therefore very likely to occur. However, even though the effects of bulk lead are well known, there is limited knowledge regarding impact of Pb NPs inhalation. This study focused on acute and subchronic exposures to lead oxide nanoparticles (PbO NPs). Mice were exposed to PbO NPs in whole body inhalation chambers for 4–72 h in acute experiment (4.05 × 106 PbO NPs/cm3), and for 1–11 weeks in subchronic experiment (3.83 × 105 particles/cm3 in lower and 1.93 × 106 particles/cm3 in higher exposure group). Presence of NPs was confirmed in all studied organs, including brain, which is very important considering lead neurotoxicity. Lead concentration gradually increased in all tissues depending on the exposure concentration and duration. The most burdened organs were lung and kidney, however liver and brain also showed significant increase of lead concentration during exposure. Histological analysis documented numerous morphological alterations and tissue damage, mainly in lung, but also in liver. Mild pathological changes were observed also in kidney and brain. Levels of glutathione (reduced and oxidized) were modulated mainly in lung in both, acute and subchronic exposures. Increase of lipid peroxidation was observed in kidney after acute exposure. This study characterized impacts of short to longer-term inhalation exposure, proved transport of PbO NPs to secondary organs, documented time and concentration dependent gradual increase of Pb concentration and histopathological damage in tissues.