Variations of manganese in the eggs of the Norway lobster, Nephrops norvegicus (L.)

The Norway lobster, Nephrops norvegicus, lives on sediments rich in manganese (Mn). Temporal fluctuations of Mn in the eggs of N. norvegicus was investigated, both in the field and in laboratory experiments. Female gonads and external eggs of different developmental stages were measured for Mn. The Mn concentration during oocyte maturation and throughout most of the embryogenesis (after fertilisation) remained stable around 5 μg Mn g(-1) dry wt. egg. At the end of the embryonic development (about 6 months after fertilisation) the Mn concentration of the egg started to increase and had at the time of hatching reached concentrations of 120 μg Mn g(-1) dry wt. egg. The egg shell was at this stage highly permeable and Mn was taken up by the embryo and egg shell in equal amounts.     

Manganese induced apoptosis in haematopoietic cells of Nephrops norvegicus (L.)

Manganese (Mn) is highly abundant as MnO2 in marine sediments. During hypoxia in bottom waters, the reduced bioavailable fraction of manganese, Mn2+, increases. Thereby, Norway lobster, Nephrops norvegicus, can experience concentrations up to 1000 times normoxic levels. A previous study has shown that exposure to a realistic concentration of 20 mg l(-1) of Mn for 10 days reduced the number of circulating haemocytes in N. norvegicus significantly. Here we aimed to investigate if apoptosis contributes to the Mn-induced haemocytopenia, with the overall hypothesis that Mn induces apoptosis in a time and concentration dependent manner. N. norvegicus were exposed to Mn (0, 5, 10 and 20 mg l(-1)) for 5 and 10 days. After 5 days of exposure the total haemocyte counts were not affected. However, after 10 days there was a gradual decrease in cell numbers, reaching a reduction by 44% when the animals were exposed to 20 mg Mn l(-1). Apoptosis in cells, released from the haematopoietic tissue, was investigated by using TUNEL assay, which detects specific DNA strand breaks. The fraction of apoptotic cells gradually increased from 2.5% in un-exposed lobsters to 15% in those exposed to 20 mg l(-1) but there was no difference related to the exposure time. A gradual increase of apoptosis was further confirmed by electrophoretic DNA-ladder formation, however to a lower extent in lobsters exposed during 5 days. Cell viability, determined by metabolic activity and cell membrane integrity, was not reduced, indicating that apoptosis rather than necrosis caused reduced number of haemocytes. It was concluded that apoptosis seemed to increase already after 5 days of 5 mg l(-1) of Mn-exposure, although exposure for 10 days was required before it was reflected in the haemocyte numbers. Reduced numbers of haemocytes may increase the prevalence for infections in N. norvegicus in their natural habitat.     

Bioaccumulation of cadmium in an experimental aquatic food chain involving phytoplankton (Chlorella vulgaris), zooplankton (Moina macrocopa), and the predatory catfish Clarias macrocephalus x C. gariepinus

The accumulation of cadmium (Cd) was studied in an experimental aquatic food chain involving the phytoplankton Chlorella vulgaris as the primary producer, the zooplankton Moina macrocopa as the primary consumer, and the catfish Clarias macrocephalus x Clarias gariepinus as the secondary consumer. C. vulgaris was first exposed to Cd solutions at 0.00, 0.35, and 3.50 mg l(-1), referred to as control group and experimental groups 1 and 2, respectively. Subsequently, each group was fed to three corresponding groups of M. macrocopa. Finally, three groups of catfish were fed these corresponding groups of M. macrocopa. After C. vulgaris was exposed to 3.50 mg l(-1) Cd (experimental group 2), the residual Cd in solution was only 4.01 microg l(-1), lower than the maximum allowable limit of Cd in natural water sources (5 microg l(-1)). Cd concentrations in C. vulgaris were 0.01+/-0.00 microg g(-1) (dry wt) in the control group, 194+/-1.80 microg g(-1) (dry wt) in experimental group 1, and 1140+/-20.06 microg g(-1) (dry wt) in experimental group 2. The Cd concentrations in M. macrocopa were 0.01+/-0.00 microg g(-1) (dry wt) in the control group, 16.48+/-2.23 microg g(-1) (dry wt) in experimental group 1, and 56.6+/-3.23 microg g(-1) (dry wt) in experimental group 2. The Cd concentrations in catfish muscle increased with increasing Cd concentrations in the food. After 60 days of fish culture, the mean concentrations of Cd in fish muscle were 0.01+/-0.00 microg g(-1) (dry wt) in the control group, 0.61+/-0.02 microg g(-1) (dry wt) in experimental group 1 and 1.04+/-0.06 microg g(-1) (dry wt) in experimental group 2. Cd concentration in fish muscle of experimental group 2 was equal to the permissible limit. Cd accumulation affected fish growth: at the end of the study, the mean fresh weight (12.81 g) of catfish in the control group, was significantly higher than those experimental group 1 (10.43 g) and experimental group 2 (10.00 g). The results showed that the measurement of Cd concentration in water does not necessarily give a measure of the safety of aquatic organisms as human food. Hence, heavy metal contamination is a matter for concern when organisms are harvested, for fish and human consumption, from natural water sources.     

Effects of manganese on chemically induced food search behaviour of the Norway lobster, Nephrops norvegicus (L.)

The decapod Norway lobster, Nephrops norvegicus (L.), lives on muddy sediments rich in manganese (Mn). In hypoxic conditions, manganese is reduced and released from the sediment, so increased concentrations of dissolved Mn(2+) become bioavailable. In excess, manganese acts as a neurotoxin and may inhibit vital functions of benthic organisms, such as muscle contraction. We investigated in a laboratory flume experiment, the effect of environmentally realistic concentrations of manganese (0.1 and 0.2mM for 12 days) on the food search behaviour of N. norvegicus. We found that lobsters exposed to manganese had a more than doubled reaction time to food odour stimuli compared to the controls (p<0.05). In addition, manganese exposure reduced the number of N. norvegicus reaching the food stimuli source. Compared to the controls where 86% reached the stimuli source, only about half of the lobsters exposed to 0.1mM Mn and one-third of the lobsters exposed to 0.2mM Mn reached the stimuli source (p<0.05 and 0.001, respectively). There was no significant difference between treatments in the number of lobsters leaving their shelter or in the time from reaction until leaving the shelter and there was no difference in search time for those animals that eventually did locate the stimuli source. This study shows that environmentally realistic manganese concentrations affect parts of the food search behaviour of N. norvegicus, likely due to impaired chemosensory ability or reduced motivation for feeding. Thus, the ability of N. norvegicus to detect and find food can be reduced in areas with high manganese concentrations, with possible consequences on individual and population levels.     

Response of spinach (Spinacea oleracea) to the added fluoride in an alkaline soil

The influence of soil contamination by inorganic fluoride (NaF) on the uptake and accumulation of fluoride in the shoot and root of spinach (Spinacea oleracea) was investigated in pot experiment under controlled conditions. The soluble fluoride in soil varied between 2.57mgkg(-1) soil and 16.44mgkg(-1) soil in the treatment range of 0-800mgNaFkg(-1) soil. It was found that the concentration of the total fluoride in shoot and root varied between 23.5mgkg(-1) dry wt. (control) and 219.8mgkg(-1) dry wt. (at 800mgNaFkg(-1) soil). The fluoride concentration in shoot and root showed a linear trend. At the added fluoride concentration beyond 200mgNaFkg(-1) of soil, the spinach root retained more fluoride than shoot. In the treatment range 0-800mgNaFkg(-1) soil, the water labile fluoride in the juice varied from 0.32 to 0.78ppm in shoot and 1.03 to 2.79ppm in the root. No visible symptom of phyto-toxicity was noticed with the treatment from 0 to 800mgNaFkg(-1) soil. It was inferred from this study that spinach (S. oleracea) accumulates fluoride at tissues level and has a distinct mechanism of partitioning of water labile fluoride and total fluoride in the tissues.     

Mucoadhesive fenretinide patches for site-specific chemoprevention of oral cancer: enhancement of oral mucosal permeation of fenretinide by coincorporation of propylene glycol and menthol

The objective of this study was to enhance oral mucosal permeation of fenretinide by coincorporation of propylene glycol (PG) and menthol in fenretinide/Eudragit RL PO mucoadhesive patches. Fenretinide is an extremely hydrophobic chemopreventive compound with poor tissue permeability. Coincorporation of 5-10 wt % PG (mean J(s) = 16-23 μg cm?2 h?1; 158-171 μg of fenretinide/g of tissue) or 1-10 wt % PG + 5 wt % menthol (mean J(s) = 18-40 μg cm?2 h?1; 172-241 μg of fenretinide/g of tissue) in fenretinide/Eudragit RL PO patches led to significant ex vivo fenretinide permeation enhancement (p < 0.001). Addition of PG above 2.5 wt % in the patch resulted in significant cellular swelling in the buccal mucosal tissues. These alterations were ameliorated by combining both enhancers and reducing PG level. After buccal administration of patches in rabbits, in vivo permeation of fenretinide across the oral mucosa was greater (～43 μg fenretinide/g tissue) from patches that contained optimized permeation enhancer content (2.5 wt % PG + 5 wt % menthol) relative to permeation obtained from enhancer-free patch (～17 μg fenretinide/g tissue) (p < 0.001). In vitro and in vivo release of fenretinide from patch was not significantly increased by coincorporation of permeation enhancers, indicating that mass transfer across the tissue, and not the patch, largely determined the permeation rate control in vivo. As a result of its improved permeation and its lack of deleterious local effects, the mucoadhesive fenretinide patch coincorporated with 2.5 wt % PG + 5 wt % menthol represents an important step in the further preclinical evaluation of oral site-specific chemoprevention strategies with fenretinide.     

Persistence of cyclic peptide toxins in dried Microcystis aeruginosa crusts from lake Mokoan,Australia

Dried Microcystis aeruginosa Kuetzing emend. Elenkin crusts estimated to be 5–6 months old from the shore of Lake Mokoan were toxic by mouse bioassay (LD₁₀₀ 100–140 mg dry wt/kg mouse). Fresh bloom material from the lake was also highly toxic (LD₁₀₀ 25–35 mg dry wt/kg mouse). Microcystin high performance liquid chromatography (HPLC) profiles of the crust and fresh material were very similar, with 24 compounds having UV spectra consistent with microcystin LR. Five of the major microcystins were purified and analysed by electrospray/mass spectrometry. The molecular weights of these microcystins [910, 924, 982, 982 (two compounds), and 986] do not correspond with known microcystins. All five compounds were hepatotoxic to mice with LD₁₀₀ values ranging from 85 to 140 μg microcystin/kg mouse). Total microcystin contents (expressed as microcystin LR aquivalents) determined by HPLC correlated with the mouse bioassay analyses (crust 2.1 μg microcystin/mg dry wt; fresh 4.1 μg microcystin/mg dry wt). These results suggest that microcystin is protected from degradation while encapsulated within the dried Microcystis crusts. Leaching experiments demonstrated that re-wetting of the crust material leads to rapid release of microcystins into the surrounding water. These observations have important management implications for lakes and reservoirs where crusts of cyanobacterial material form on the shoreline. © by John Wiley & Sons, Inc.     

Excursions of Intake above ADI: Case Study on Cadmium

High oral intake of cadmium via food or drink in a single dose by humans gives rise to vomiting, abdominal pain, and diarrhea. Concentrations of cadmium in drinks giving rise to such symptoms have been 16 mg/liter and higher corresponding to doses of 3 mg and higher. Longer term intakes of food (rice) with concentrations around 1 mg/kg corresponding to daily intakes of 600 μg have given rise to some less pronounced symptoms including signs of malabsorption. Reproductive and developmental effects have been observed in animal experiments at oral and other exposures. The present provisional tolerable weekly intake (PTWI) for Cd is 500 μg (a weekly intake of 7 μg/kg body wt), corresponding to a daily intake of 70 μg or 1 μg per kg body wt. Recent data demonstrating renal dysfunction in humans at even lower lifelong oral exposures indicate that the PTWI needs to be lowered in the future. An estimated lowest-observed-adverse-effect level (LOAEL) for symptoms from the gastrointestinal tract in humans after intake of a single oral dose is 43 μg/kg body wt. If a safety factor of 3–10 is used based on LOAEL, a tolerable single dose would be 0.3–1 mg (4 to 14 μg/kg body wt). For longer time exposures (months–a few years) daily intakes of 200 μg (3 μg/kg body wt) may be tolerated without obvious gastrointestinal symptoms or signs. At present, there is no convincing human evidence that such doses can cause reproductive or developmental effects, but since such effects have been reported in animals, it may be advisable not to exceed a daily intake of 1 μg/kg body wt for such potentially sensitive subsections of the population as children and women who are pregnant or lactating. Any excursions above the PTWI need to be compensated for by a corresponding period with intake below the PTWI in order for the cumulative dose to be low enough to avoid the long-term effects of cadmium on the kidney.     

Manganese induced immune suppression of the lobster, Nephrops norvegicus

Manganese (Mn) is one of the most abundant elements on earth, particularly in the soft bottom sediments of the oceans. As a micronutrient Mn is essential in the metabolic processes of organisms. However, at high concentrations the metal becomes a neurotoxin with well-documented effects. As a consequence of euthrophication, manganese is released from bottom sediments of coastal areas and the Norway lobsters, Nephrops norvegicus, can experience high levels of bioavailable Mn(2+). Here, we present the first report showing that Mn also affects several fundamental processes in the mobilisation and activation of immunoactive haemocytes. When N. norvegicus was exposed to a realistic [Mn(2+)] of 20 mg l(-1) for 10 days 24.1 microg ml(-1) was recorded in the haemolymph. At this concentration the total haemocyte count was reduced by ca. 60%. By using BrdU as a tracer for cell division, it was shown that the proliferation rate in the haematopoietic tissue did not increase, despite the haemocytepenia. A gene coding for a Runt-domain protein, known to be involved in maturation of immune active haemocytes in a variety of organisms, was identified also in haemocytes of N. norvegicus. The expression of this gene was >40% lower in the Mn-exposed lobsters as judged by using a cDNA probe and the in situ hybridisation technique. In response to non-self molecules, like lipopolysaccharide (LPS), the granular haemocytes of arthropods are known to degranulate and thereby release and activate the prophenoloxidase system, necessary for their immune defence. A degranulation assay, tested on isolated granular haemocytes, showed about 75% lower activity in the Mn-exposed lobsters than that for the unexposed. Furthermore, using an enzymatic assay, the activation per se of prophenoloxidase by LPS was found blocked in the Mn-exposed lobsters. Taken together, these results show that Mn exposure suppressed fundamental immune mechanisms of Norway lobsters. This identifies a potential harm that also exists for other organisms and should be considered when increasing the distribution of bioavailable Mn, as has been done through recently introduced applications of the metal.     

Acute toxicity and bioaccumulation of arsenic in tilapia (Oreochromis mossambicus) from a blackfoot disease area in Taiwan

The general objective of our work was to determine the acute toxicity and bioaccumulation of arsenic (As) in tilapia (Oreochromis mossambicus) from the blackfoot disease (BFD) area in Taiwan. The average concentration of As in pond water ranged from 17.8 to 49 microg L(-1). Acute toxicity tests showed that the As concentration that caused toxicity to tilapia ranged from 69 060 microg As L(-1), in the 24-h toxicity test, to 28 680 microg As L(-1), in the 96-h toxicity test. We measured As concentrations in various tissues of tilapia to identify the affinities of tissues for As. Significant correlations were found among the As concentrations in all tissues. The highest bioconcentration factor (BCF) was found in the intestine (maximum value: 2270). The order of BCFs was: intestine > stomach > liver approximately gill > muscle. Arsenic concentrations in all tissues were allometric, negatively correlating with fish body weight [r(2) = 0.63 +/- 0.045 (mean +/- SE), p < 0.05]. Our results also revealed that As concentrations in muscle tissue were positively correlated with As accumulation in the viscera (r(2) = 0.85, p < 0.05). Significantly higher concentrations of As were obtained in the viscera of tilapia [12.65 +/- 10.17 microg g(-1) dry wt (mean +/- SD)] than in the muscle tissue (3.55 +/- 0.42 microg g(-1) dry wt). Our results suggest that a simple way of reducing the health risk associated with consuming tilapia is to trim and cook the fish properly, that is, removing the viscera of tilapia can greatly reduce the amount of As ingested and consequently reduce the health risks.     

Action of excess calcium ions in the brain on motivated feeding in the rat: attenuation by pharmacological antagonists

The intraventricular infusion of 5 μl of physiological solution in which the concentration of calcium ions was adjusted from 1.26 mM to 151.2 mM caused spontaneous feeding and drinking in the satiated rat. The ingestive response was observed both when pellets and water freely available, in which case food consumption exhibited a dose-response relationship increasing with the millimolarity of calcium in the infusate, and when operant responses at FR 2 or FR 10 were required for food reward. The rate of bar pressing by an animal at these two schedules of reinforcement did not differ from that observed when the same animal was deprived of food and water for 23 hr. The alpha-adrenergic antagonist, phentolamine (16 μg, 32 μg), did not appreciably attenuate Ca²⁺-induced feeding during either ad lib or motivated conditions, though these doses drastically reduced food intake induced by the intraventricular infusion of 10 μg norepinephrine HCl. The pre-infusion of a number of additional blocking agents had variable yet consistent effects upon Ca²⁺-induced feeding; hexamethonium (8 μg, 25 μg), propranolol (6 μg, 28 μg) and methysergide (8 μg) each caused a statistically significant enhancement in the magnitude of the Ca²⁺ response while atropine (6 μg, 15 μg) significantly attenuated feeding after Ca²⁺. This suggests an interaction between a number of pharmacologically distinct fiber pathways that may mediate the Ca²⁺ feeding response. The modulation of feeding by Ca²⁺ ions supports an ionic theory for the regulation of body weight in the rat, in which neurons that are involved in ingestive responses and which pass through the hypothalamus and other structures along the ventricular lumen are selectively sensitive to the concentration of calcium in the extracellular fluid.     

Liposomes as an alternative delivery system for investigating dietary metal toxicity to Daphnia magna

Dietary metal toxicity studies with invertebrates such as Daphnia magna are often performed using metal-contaminated algae as a food source. A drawback of this approach is that it is difficult to distinguish between the direct toxicity of the metal and indirect effects caused by a reduced essential nutrient content in the metal-contaminated diet, due to prior exposure of the algae to the metal. This hampers the study of the mechanisms of dietary metal toxicity in filter-feeding freshwater invertebrates. The aim of the present study was to develop a technique for producing metal-contaminated liposomes as an alternative delivery system of dietary metals. These liposomes are not vulnerable to metal-induced shifts in nutrient quality. Liposomes were prepared by the hydration of phosphatidylcholine in media containing either 0 (control) or 50mg Ni/L. The liposomes had average diameters of 19.31 (control) and 10.48 μm (Ni-laden), i.e., a size appropriate for ingestion by D. magna. The liposome particles were then mixed with uncontaminated green algae in a 1/10 ratio (on a dry wt. basis) to make up two diets that differed in Ni content (i.e., 2.0 μg Ni/g dry wt. in the control and 144.2 μg Ni/g dry wt. in the Ni-contaminated diet, respectively). This diet was then fed to D. magna during a 21-day chronic bioassay. The experiment showed that the Ni content and the size distribution of the liposomes were stable for at least 7 days. Also the use of phosphatidylcholine as a liposome component did not affect the reproduction of the daphnids. Exposure to increased level of dietary Ni resulted in 100% mortality after 14 days of exposure and in an increased whole-body Ni concentration in D. magna of 14.9 and 20.4 μg Ni/g dry wt. after 7 and 14 days of exposure, respectively. The Ni-exposed daphnids also exhibited a reduced size (i.e., 30% smaller than the control) after 7 days and a completely halted growth between day 7 and day 14. In terms of reproduction, the size of the first brood (number of juveniles) of the Ni-exposed daphnids was significantly reduced (by 85%) compared to the control. None of the Ni-exposed individuals were able to produce a second brood before dying. The algal ingestion rate - after correction for the indirect effect of a reduced size - was increased (by 68%) by dietary Ni after 6 days of exposure compared to the control, but was severely reduced (by 80% compared to the control) after 13 days. These data suggest that an inhibition of the ingestion process may have contributed to the observed effects of dietary Ni on growth and reproduction beyond 6 days of exposure, although the involvement of other mechanisms cannot be excluded. The mechanism(s) which led to the reduced growth during the first week of exposure remain unclear, although inhibition of the ingestion process can likely be excluded here as an explanation. Overall, this paper demonstrates, using this new method of delivering dietary Ni via liposome carriers and thus excluding potential diet quality shifts, that dietary Ni can indeed induce toxic effects in D. magna. This method may therefore be a promising tool to help further elucidate the mechanisms of dietary metal toxicity to filter-feeding invertebrates.     

Potentiometric determination of polyether ionophores growth promotors in animal feed preparations

The construction and electrochemical response characteristics of four polyvinylchloride (PVC) membrane sensors for the determination of monensin (MN) and salinomycin (SL) were described. The membranes were prepared using 1 wt.% drug, 44 wt.% nitrophenyl octyl ether, 53 wt.% PVC and 2 wt.% lipophilic additive which is the anionic potassium tetra (4-chlorophenyl) borate in sensors 1 and 2 and the cationic nicklo-phenanthroline in sensors 3 and 4. Sensor 1 and sensor 2 show linear responses over concentration range of 10(-3)-10(-5) M drug with cationic slopes of 52.3 and 54.1 mV per concentration decade, respectively. On the other hand sensor 3 and sensor 4 show linear responses over concentration range 10(-4)-10(-5) M drug with anionic slopes of 28.1 and 29.7 mV per concentration decade, respectively. The 4 sensors were successfully applied to the determination of MN and SL in their pharmaceutical products (Premix) with average recoveries of 98.4-100.4+/-2.41-1.97% for sensors 1 and 2 and 97.6-98.8+/-3.16-3.07% for sensors 3 and 4. The obtained results were compared reasonably well with the data obtained using the USP method (2000). The proposed sensors were also applied for the direct determination of both drugs in animal feed preparations without prior treatment in low levels; 10-1000 microg per 5 g feed. The sensors were successfully used to follow up the drugs concentration in the presence of other growth promotors, other antibiotics and the coexisting fatty acids.     

Effects of flurazepam on amino acid-evoked responses recorded from the lobster muscle and the frog spinal cord.

The effects of flurazepam hydrochloride on GABA- or glutamate-evoked responses recorded from the lobster muscle fibre and the frog isolated spinal cord were studied using electrophysiological techniques. On lobster muscle flurazepam (up to 100 μM) reversibly antagonized responses to bath-applied or iontophoretically-applied glutamate without any effect on GABA responses. Higher concentrations of flurazepam (>200 μM) sometimes increased the resting membrane conductance, an effect different from that of GABA in being insensitive to picrotoxin (0.5 μM). In the tetrodotoxin (TTX)-treated frog spinal cord flurazepam (5 μM) reversibly antagonized both glutamate- and GABA-evoked dorsal root depolarizations although a smaller dose (2.5 μM) clearly potentiated the action of GABA. Spontaneous fluctuations of the dorsal root d.c. level in the presence of flurazepam were also observed. It is suggested that flurazepam antagonized amino acid responses by blocking receptor-activated Na⁺ channels on the postsynaptic membranes. However, the potentiation of GABA action on the frog spinal cord may have involved either release of endogenous GABA or “sensitization” of spinal receptors to GABA, an action easily obscured by the predominant antagonistic effect of flurazepam at higher concentrations.     

In vitro human epidermal penetration of 1-bromopropane

1-Bromopropane (1-BP; CAS number 106-94-5), also known as n-propyl bromide, is a halogenated short-chain alkane used as an organic solvent with numerous commercial and industrial applications, including garment dry cleaning and vapor degreasing of metals. The purpose of this study was to determine the dermal absorption characteristics and corrosivity of 1-BP. Heat-separated human epidermal membranes were mounted on static diffusion cells. Different exposure scenarios were studied (infinite dose, finite dose, and transient exposure) using neat 1-BP and saturated aqueous solution as donor. Steady-state fluxes for infinite-dose neat 1-BP exposure averaged 625 to 960 μg cm(-2) h(-1). The finite-dose (10 μl/cm(2) = 13.5 mg/cm(2)) unoccluded donor resulted in penetration of <0.2% of the applied dose (22 μg/cm(2)). A 10-min transient exposure to infinite dose resulted in total penetration of 179 μg/cm(2). Steady-state 1-BP fluxes from neat application of a commercial dry cleaning solvent were similar (441 to 722 μg cm(-2) h(-1)). The permeability coefficient of 1-BP in water vehicle was 0.257 ± 0.141 cm/h. The absorption potential of 1-BP following dermal exposure is dependent upon the type and duration of exposure. Donor losses due to evaporation were approximately 500-fold greater than dermal absorption flux; evaporation flux was 420 mg cm(-2) h(-1). 1-BP is cytotoxic but not corrosive, based on results from a cultured reconstructed human epidermal model (EpiDerm Skin Corrosivity Test).     

Accumulation of biologically active furanocoumarins in agitated cultures of Ruta graveolens L. and Ruta graveolens ssp. divaricata (Tenore) Gams

This study was designed to investigate the dynamics of accumulation of linear furanocoumarins (psoralen, bergapten, xanthotoxin, isopimpinellin, imperatorin) and their biogenetic precursor, umbelliferone, in agitated cultures of Ruta graveolens L. and Ruta graveolens ssp. divaricata (Tenore) Gams during 6-week growth cycles. The metabolites under study were almost exclusively accumulated in the cultured biomass. The total content of all metabolites increased 4.8- and 2.0-fold, in R. graveolens and R. graveolens ssp. divaricata cultures, respectively. Xanthotoxin and bergapten, which are the most important therapeutic compounds, were the dominating metabolites in cultures of both plants. The maximum content of xanthotoxin (25.0 mg/100 g dry wt.) and bergapten (18.4 mg/100 g dry wt) and the maximum content of all metabolites (64.0 mg/100 g dry wt) in R. graveolens ssp. divaricata callus obtained on the 35th culture day were relatively low. However, maximum contents of xanthotoxin (136.8 mg/100 g dry wt), bergapten (210.4 mg/100 g dry wt.) and isopimpinellin (96.7 mg/100 g dry wt), and total content of all metabolites in R. graveolens shoots (520.8 mg/100 g dry wt) obtained on the 42nd culture day are interesting from a practical point of view.     

Mercury transfer in a subtropical coastal lagoon food web (SE Gulf of California) under two contrasting climatic conditions

Mercury (Hg) transference through an established and defined food web from an urbanized subtropical coastal lagoon (SE Gulf of California) was examined by using carbon and nitrogen stable isotope and Hg analyses. Concentrations of Hg in seawater (0.045-0.078 μg L(-1)), predominantly incorporated to the particulate fraction (60% of total), were lower than those found in highly contaminated coastal areas around the world (0.18-16 μg L(-1)). Although levels of Hg available for the biota (e.g., associated to the reactive and pyrite fractions; 0.11-0.15 μg g(-1)) were within of effects range-low (ER-L, 0.15-0.71 μg g(-1)) these concentrations represented from 46.6 to 67.0% of the total Hg pool that may be transferred through food web. The sequence of bio-accumulation of Hg in studied species was according with their functional guild: tertiary consumer (0.85-1.15 μg g(-1)) > secondary consumers (0.084-0.168 μg g(-1)) > primary consumers (0.014-0.160 μg g(-1)) > primary producers (0.016-0.056 μg g(-1)). Hg concentrations increased for each successive trophic position estimate through δ(15) N isotopic values, although these relationships showed differences between dry and rainy seasons (R(2) = 0.37 and R(2) = 0.39 between δ(15) N vs. Hg in organisms, respectively). We concluded that Hg is being positively transferred (biomagnification factor >1) through the studied food web probably enhanced by the favorable environmental conditions for Hg-methylation (e.g., fine sediments rich in organic matter, and environmental conditions changes of the redox, pH, and temperature) found in this urbanized coastal water, however, the study of processes methlylation and biomagnifcation of Hg need further investigations.     

Effect of chronic treatment with an estrogen-progestogen combination on beta adrenergic-induced thirst.

Rats chronically administered various doses of the estrogenic agent, ethinyl estradiol, combined with the progestogen, norethynodrel, showed an estrogen-related, dose-dependent decrease in water intake in response to acute SC administration of the beta adrenergic agonist, isoproterenol (200 μg/kg of body wt). Conversely, rats receiving a constant dose of ethinyl estradiol (16 μg/kg/day) combined with norethynodrel (233 μg/kg/day) for 7 weeks drank significantly less water in response to graded doses of isoproterenol (10, 25, 50, 100 and 200 μg/kg) than controls. The slope, but not the intercept, of the regression line relating water intake to dose of isoproterenol administered was reduced significantly in the steroid-treated group. Furthermore, water intake of the treated group in response to acute administration of either epinephrine (1 mg/kg) or serotonin (1 mg/kg) was significantly less than control. These data suggest that chronic treatment with ethinyl estradiol, combined with norethynodrel, reduced beta-adrenergic responsiveness. In addition, rats treated with the same combination of ethinyl estradiol and norethynodrel showed a reduced drinking response to an osmotic thirst stimulus, i.e., IP load of 1 M NaCl solution at 1% of body wt. The fact that the response to both types of thirst stimuli was attenuated in rats receiving an estrogen combined with a progestogen suggests the possibility that treatment may have affected thirst mechanisms at the level of the central nervous system.     

The carcinogenicity of trichloroethylene and its metabolites, trichloroacetic acid and dichloroacetic acid, in mouse liver

Trichloroethylene (TCE) has previously been shown to be carcinogenic in mouse liver when administered by daily gavage in corn oil. The metabolism of TCE results, in part, in the formation of trichloroacetic acid (TCA) as a major metabolite and dichloroacetic acid (DCA) as a minor metabolite. These chlorinated acetic acids have not been shown to be genotoxic, although they have been shown to induce peroxisome proliferation. Therefore, we determined the ability they have been shown to induce peroxisome proliferation. Therefore, we determined the ability of TCE, TCA, or DCA to act as tumor promoters in mouse liver. Male B6C3F1 mice were administered intraperitoneally 0, 2.5, or 10 micrograms/g body wt ethylnitrosourea (ENU) on Day 15 of age. At 28 days of age, the mice were placed on drinking water containing either TCE (3 or 40 mg/liter), TCA (2 or 5 g/liter), or DCA (2 or 5 g/liter). All drinking waters were neutralized with NaOH to a final pH of 6.5-7.5. The animals were killed after 61 weeks of exposure to the treated drinking water (65 weeks of age). Both DCA and TCA at a concentration of 5 g/liter were carcinogenic without prior initiation with ENU, resulting in hepatocellular carcinomas in 81 and 32% of the animals, respectively. DCA and TCA also increased the incidence of animals with adenomas and the number of adenomas/animal in those animals that were not initiated with ENU. While 2.5 micrograms/g body wt ENU followed by NaCl in the drinking water resulted in only 5% of the animals with hepatocellular carcinomas, 2.5 micrograms/g body wt ENU followed with 2 or 5 g/liter DCA resulted in a 66 or 78% incidence of carcinoma, respectively, or, followed with 2 or 5 g/liter TCA, resulted in a 48% incidence at either concentration. None of the untreated animals had hepatocellular carcinomas. Therefore our results demonstrate that DCA and TCA are complete hepatocarcinogens in B6C3F1 mice.     

Multivariate approach in the optimization procedures for the direct determination of manganese in serum samples by graphite furnace atomic absorption spectrometry

A method for direct determination of manganese (Mn) in human serum by graphite furnace atomic absorption spectrometry (GFAAS) was proposed in this work. The samples were only diluted 1:4 with nitric acid 1% (v/v) and Triton(?) X-100 0.1% (v/v). The optimization of the instrumental conditions was made using multivariate approach. A factorial design (2(3)) was employed to investigate the tendency of the most intense absorbance signal. The pyrolysis and atomization temperatures and the use of modifier were available and only the parameter modifier use did not have a significant effect on the response. A Center Composed Design (CCD) presented best temperatures of 430 °C and 2568 °C for pyrolysis and atomization, respectively. The method allowed the determination of manganese with a curve varying from 0.7 to 3.3 μg/L. Recovery studies in three concentration levels (n=7 for each level) presented results from 98 ± 5 to 102 ± 7 %. The detection limit was 0.2 μg/L, the quantifying limit was 0.7 μg/L, and the characteristic mass, 1.3 ± 0.2 pg. Intra- and interassay studies showed coefficients of variation of 4.7-7.0% (n=21) and 6-8%(n=63), respectively. The method was applied for the determination of manganese in 53 samples obtaining concentrations from 3.9 to 13.7 μg/L.