Marijana Curcic

Fluoride in drinking water and dental fluorosis.

In this study we determined the fluoride content in drinking water and hair of 12-year-old schoolchildren from different Serbian municipalities, i.e. Valjevo, Veliko Gradiste, Kacarevo and Vranjska Banja. The analyses were performed using composite fluoride ion-selective electrode. Average fluoride levels were 0.10, 0.15, 0.79 and 11 ppm in well water, 0.07, 0.10, 0.17 and 0.15 ppm in tap water, 19.3, 21.5, 25.4, and 32.5 ppm in hair samples, in Valjevo, Veliko Gradiste, Kacarevo and Vranjska Banja, respectively. Correlation analysis indicated statistically significant positive relationship between fluoride in wells water and fluoride in hair, for all municipalities: correlation coefficients were 0.54 (p < 0.05), 0.89, 0.97 and 0.99 (p < 0.001), in Vranjska Banja, Valjevo, Veliko Gradiste, and Kacarevo, respectively. Positive correlation was obtained also between fluoride in tap water and hair samples in all regions under the study, with statistical significance only in Valjevo municipality, p < 0.05. Dental examination of schoolchildren confirmed dental fluorosis only in the region of Vranjska Banja. Moreover, in endemic fluorotic region of Vranjska Banja, positive and statistically significant correlations were confirmed between fluoride in well water and dental fluorosis level (r = 0.61; p < 0.01) and additionally between fluoride in hair and dental fluorosis level (0.62; p < 0.01). The primary findings from this study have shown that fluoride content in hair is highly correlated with fluoride content in drinking water and dental fluorosis level, indicating that hair may be regarded as biomaterial of high informative potential in evaluating prolonged exposure to fluorides and to individuate children at risk of fluorosis regardless of the phase of teeth eruption.

Protective role of glutathione reductase in paraquat induced neurotoxicity

Paraquat (PQ), a widely used herbicide is a well-known free radical producing agent. The mechanistic pathways of PQ neurotoxicity were examined by assessing oxidative/nitrosative stress markers. Focus was on the role of glutathione (GSH) cycle and to examine whether the pre-treatment with enzyme glutathione reductase (GR) could protect the vulnerable brain regions (VBRs) against harmful oxidative effect of PQ. The study was conducted on Wistar rats, randomly divided in five groups: intact-control group, (n = 8) and four experimental groups (n = 24). All tested compounds were administered intrastriatally (i.s.) in one single dose. The following parameters of oxidative status were measured in the striatum, hippocampus and cortex, at 30 min, 24 h and 7 days post treatment: superoxide anion radical (O₂·⁻), nitrate (NO₃⁻), malondialdehyde (MDA), superoxide dismutase (SOD), total GSH (tGSH) and its oxidized, disulfide form (GSSG) and glutathione peroxidase (GPx). Results obtained from the intact and the sham operated groups were not statistically different, confirming that invasive i.s. route of administration would not influence the reliability of results. Also, similar pattern of changes were observed between ipsi- and contra- lateral side of examined VBRs, indicating rapid spatial spreading of oxidative stress. Mortality of the animals (10%), within 24h, along with symptoms of Parkinsonism, after awakening from anesthesia for 2-3 h, were observed in the PQ group, only. Increased levels of O₂·⁻, NO₃⁻ and MDA, increased ratio of GSSG/GSH and considerably high activity of GPx were measured at 30 min after the treatment. Cytotoxic effect of PQ was documented by drastic drop of all measured parameters and extremely high peak of the ratio GSSG/GSH at 24th hrs after the PQ i.s. injection. In the GR+PQ group, markedly low activity of GPx and low content of NO₃⁻ (in striatum and cortex) were measured during whole experiment, while increase value was observed only for O₂·⁻, at 7th days. We concluded that oxidative/nitrosative stress and excitotoxicity are the most important events since the early stage of PQ induced neurotoxicity. Based on the ratio GSSG/GSH, the oxidation of GSH to GSSG is probably dominant way of GHS depletion and main reason for reduced antioxidative defense against PQ harmful oxidative effect. The GR pre-treatment resulted in the absence of Parkinsons disease-like symptoms and mortality of the rats. Additionally, oxidative/nitrosative stress did not developed, as well as almost diminished metabolism of the VBRs at 24th hours (as has been documented in the PQ group) did not occurred in the GR+PQ, suggesting a neuroprotective role for the GR in PQ induced neurotoxicity.

Relationship between fluoride intake in Serbian children living in two areas with different natural levels of fluorides and occurrence of dental fluorosis

The amount of fluoride present naturally in drinking water is highly variable, being dependent upon the individual geological environment from which the water is obtained. Chronic exposure to exceeding fluoride doses induces set of toxic effects, i.e. fluorosis. The aim of this study was to examine fluoride content in water and in the most frequently used vegetables, potato and bean, grown in two different Serbian regions, i.e. control region (Valjevo) and high naturally occurring fluoride region (Vranjska Banja), and moreover, to correlate estimated daily intake with dental fluorosis occurrence as an adverse effect of fluoride exposure of schoolchildren in Serbia. Study confirmed significant difference in fluoride content in water, potato and bean, consumed by 12-year-old children in two investigated municipalities. Results of the study indicated positive and statistically significant correlation between daily intake of fluoride and dental fluorosis level in the fluorotic municipality of Vranjska Banja (r = 0.61; p = 0.000017). Obtained relationship could be evaluated by means of binary logistic regression analysis, whereas probability for fluorosis occurrence could be predicted using the following equation: fluorosis occurence (%) = (34.852 x Cwater -12.644 x Cpotato - 9.362 x Cbean - 7.673) x 100 (Chi-Square (3) = 33.033; p < 0.001).

Combined effects of cadmium and decabrominated diphenyl ether on thyroid hormones in rats.

The aim of this study was to see how a mixture of cadmium (Cd) and decabrominated diphenyl ether (BDE209) affect thyroid function, namely thyroid-stimulating hormone (TSH), thyroxin (T4), free thyroxin (FT4), triiodothyronin (T3), and free triiodothyronin (FT3) in Wistar rats (eight per group) receiving either a single substance or their combination by gavage for 28 days. Three groups were receiving Cd alone in the doses of 2.5 mg kg-1, 7.5 mg kg-1, or 15 mg kg-1 b. w. a day, three groups were receiving BDE209 in the doses of 1000 mg kg-1, 2000 mg kg-1, or 4000 mg kg-1 b. w. a day, while nine groups were receiving different mixtures of Cd and BDE209 in these doses (3x3 design). The results have indicated that the Cd+BDE209 mixtures more potently disrupt thyroid hormone homeostasis than would be expected from these chemicals alone.

Cadmium and decabrominated diphenyl ether mixture: In vitro evaluation of cytotoxic, prooxidative and genotoxic effects.

In order to look into the combined effects of Cd and BDE-209 in vitro, this study was aimed at examining cytotoxic and genotoxic effects using the human colon carcinoma cell line (SW 480) as a biological test system as well as to determine if ROS production was one of the possible mechanisms of their mixture action. This cell line was chosen since ingestion of contaminated food/water represents an important route of exposure to both Cd and BDE-209, which is why intestinal cells are a common target for the contaminants present in food and water. Cells were treated with single Cd in concentrations of 2.5, 7.5 or 15μg Cd/mL (corresponding to 22, 67 or 134μM), single BDE-209 in concentrations of 2.5, 5 or 10μg BDE209/mL (corresponding to 2.5, 5 or 10μM), and their mixtures (design 3×3). Mixture of Cd and BDE-209 has shown clear potential to reduce the viability of SW 480 cells, as evidenced by cytotoxicity associated with ROS generation. Factorial regression models used to identify type of interaction revealed synergism related to mixture citotoxicity and additive interaction for the effect on ROS production. The results from this introductory study could contribute to the issue of possible adverse effects associated with co-exposure and body burden with two persistent environmental pollutants, Cd and BDE-209.

Fresh frozen plasma as a successful antidotal supplement in acute organophosphate poisoning.

Despite improvements to intensive care management and specific pharmacological treatments (atropine, oxime, diazepam), the mortality associated with organophosphate (OP) poisoning has not substantially decreased. The objective of this examination was to describe the role of fresh frozen plasma (FFP) in acute OP poisoning. After a deliberate ingestion of malathion, a 55-year-old male suffering from miosis, somnolence, bradycardia, muscular fasciculations, rales on auscultation, respiratory insufficiency, as well as from an inhibition of red blood cell acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BuChE), was admitted to hospital. Malathion was confirmed in a concentration of 18.01 mg L-1. Apart from supportive measures (including mechanical ventilation for four days), antidotal treatment with atropine, oxime - pralidoxime methylsulphate (ContrathionR), and diazepam was administered, along with FFP. The potentially beneficial effects of FFP therapy included a prompt increase of BuChE activity (from 926 IU L-1 to 3277 IU L-1; reference range from 7000 IU L-1 to 19000 IU L-1) and a reduction in the malathion concentration, followed by clinical recovery. Due to BuChE replacement, albumin content, and volume restitution, FFP treatment may be used as an alternative approach in patients with acute OP poisoning, especially when oximes are not available. Sažetak Unatoč napretku intenzivnog liječenja i specifične farmakološke terapije (atropin, oksim, diazepam), mortalitet u akutnim trovanjima organofosfatima (engl. Organophosphate, OP) nije se značajno smanjio. Cilj je bio upozoriti na ulogu svježe smrznute plazme (engl. Fresh frozen plasma, FFP) u liječenju akutnih otrovanja OP-ima. Nakon namjerne ingestije malationa, u bolnicu je primljen muškarac u dobi od 55 godina s miozom, somnolencijom, bradikardijom, mišićnim fascikulacijama, pukotima pri auskultaciji pluća i respiratornom insuficijencijom kao karakterističnim znacima trovanja organofosfatom, kao i inihibicijom eritrocitne acetilkolinesteraze (AChE) i plazmatske butirilkolinesteraze (BuChE). U krvi je potvrđena prisutnost malationa u koncentraciji od 18,01 mg L-1. Pored pratećih mjera (uključujući mehaničku ventilaciju tijekom četiri dana), primijenjena je antidotska terapija atropinom, oksimom pralidoksim-metilsulfatom (ContrathionR) i diazepamom, zajedno s FFP-om. Naglo povećanje aktivnosti BuChE (od 926 IU L-1 do 3277 IU L-1; referentna vrijednost od 7000 IU L-1 do 19000 IU L-1), eliminacija otrova s promptnom redukcijom koncentracije malationa, uz klinički oporavak, mogu se pripisati povoljnim učincima terapije FFP-om kao adjuvansom. FFP osigurava BuChE, povećani sadržaj albumina i nadoknadu volumena, zbog čega se može koristiti kao alternativni način liječenja akutnih trovanja OP-ima, posebno kada nam nisu dostupni oksimi.

Interactions between cadmium and decabrominated diphenyl ether on blood cells count in rats—Multiple factorial regression analysis

The objective of this study was to assess toxicity of Cd and BDE-209 mixture on haematological parameters in subacutely exposed rats and to determine the presence and type of interactions between these two chemicals using multiple factorial regression analysis. Furthermore, for the assessment of interaction type, an isobologram based methodology was applied and compared with multiple factorial regression analysis. Chemicals were given by oral gavage to the male Wistar rats weighing 200-240g for 28days. Animals were divided in 16 groups (8/group): control vehiculum group, three groups of rats were treated with 2.5, 7.5 or 15mg Cd/kg/day. These doses were chosen on the bases of literature data and reflect relatively high Cd environmental exposure, three groups of rats were treated with 1000, 2000 or 4000mg BDE-209/kg/bw/day, doses proved to induce toxic effects in rats. Furthermore, nine groups of animals were treated with different mixtures of Cd and BDE-209 containing doses of Cd and BDE-209 stated above. Blood samples were taken at the end of experiment and red blood cells, white blood cells and platelets counts were determined. For interaction assessment multiple factorial regression analysis and fitted isobologram approach were used. In this study, we focused on multiple factorial regression analysis as a method for interaction assessment. We also investigated the interactions between Cd and BDE-209 by the derived model for the description of the obtained fitted isobologram curves. Current study indicated that co-exposure to Cd and BDE-209 can result in significant decrease in RBC count, increase in WBC count and decrease in PLT count, when compared with controls. Multiple factorial regression analysis used for the assessment of interactions type between Cd and BDE-209 indicated synergism for the effect on RBC count and no interactions i.e. additivity for the effects on WBC and PLT counts. On the other hand, isobologram based approach showed slight antagonism for the effects on RBC and WBC while no interactions were proved for the joint effect on PLT count. These results confirm that the assessment of interactions between chemicals in the mixture greatly depends on the concept or method used for this evaluation.

Oxidative Stress Markers in GnRH AgonistaAnd Antagonist Protocols in IVF

Summary Background: Our aim was to study the effect of GnRH agonist and antagonist protocols of ovarian stimulation on oxidative stress parameters in serum and the influence of oxidative stress parameters change on the outcome of IVF cycles. Methods: This prospective study included 82 patients who un der went IVF procedures. We determined SOD, MDA and SH groups in serum. Serum samples were obtained between the second and fourth day of the cycle and on the day of HCG administration during ovarian stimulation. Results: Patients were divided into two groups depending on the protocol of stimulation. The mean total and mature oocytes number and number of fertilized oocytes were higher in GnRH agonist group. There was no significant difference in biochemical pregnancy, miscarriage and live -birth rate in both groups. Mean serum SOD was significantly lower, while mean serum MDA and SH groups were significantly higher after ovarian stimulation. Delivery rate was higher in patients without OS while miscarriage rate was higher in patients with OS. Conclusions: Our study confirmed that there is a difference in the concentration of oxidative stress parameters before and after ovarian stimulation. IVF outcome is better in patients without OS after ovarian stimulation. However, the protocol of ovarian stimulation is neither associated with a change in oxidative stress parameters nor with the outcome of ART procedures.

Overview of Cadmium Thyroid Disrupting Effects and Mechanisms

Humans are exposed to a significant number of chemicals that are suspected to produce disturbances in hormone homeostasis. Hence, in recent decades, there has been a growing interest in endocrine disruptive chemicals. One of the alleged thyroid disrupting substances is cadmium (Cd), a ubiquitous toxic metal shown to act as a thyroid disruptor and carcinogen in both animals and humans. Multiple PubMed searches with core keywords were performed to identify and evaluate appropriate studies which revealed literature suggesting evidence for the link between exposure to Cd and histological and metabolic changes in the thyroid gland. Furthermore, Cd influence on thyroid homeostasis at the peripheral level has also been hypothesized. Both in vivo and in vitro studies revealed that a Cd exposure at environmentally relevant concentrations results in biphasic Cd dose-thyroid response relationships. Development of thyroid tumors following exposure to Cd has been studied mainly using in vitro methodologies. In the thyroid, Cd has been shown to activate or stimulate the activity of various factors, leading to increased cell proliferation and a reduction in normal apoptotic activity. Evidence establishing the association between Cd and thyroid disruption remains ambiguous, with further studies needed to elucidate the issue and improve our understanding of Cd-mediated effects on the thyroid gland.

Dose-response modeling of reactivating potency of oximes K027 and K203 against a direct acetylcholinesterase inhibitor in rat erythrocytes

Inhibition of acethylcholinesterase (AChE) as a key molecular event induced by organophosphate (OP) pesticides and nerve agents presents a human health concern. In efficacy testing of experimental oximes, potential antidotes in OP poisoning, reactivation of OP-inhibited AChE is used as specific endpoint. However, according to our best knowledge, so far oximes have not been quantitatively evaluated by comprehensive benchmark dose (BMD) approach, that would improve both identification and quantification of the effect and allow more rigorous comparison of efficacies. Thus, we have examined in vivo dose-response relationship for two promising experimental oximes, K203 and K027, concerning reactivation of erythrocyte AChE inhibited by dichlorvos (DDVP). Groups of Wistar rats were treated with six different doses of oximes (i.m) immediately after DDVP challenge (s.c) and AChE was measured 60 min later. Dose-response modeling was done by PROAST software 65.5 (RIVM, The Nederlands). BMD-covariate method resulted in four-parameter model from both exponential and Hill model families as the best estimate of relationship between AChE activity and oxime dose, with potency parameter being oxime-dependent. Oxime K027 was shown to be 1.929-fold more potent considering that 58% increase in AChE activity was achived with the dose BMD58-K027 = 52 μmol/kg in contrast to BMD58-K203 = 100 μmol/kg.