Toxic and essential metal concentrations in four tissues of red deer (Cervus elaphus) from Baranja, Croatia.

Kidney (n = 297), liver (n = 52), jawbone (n = 80) and muscle (n = 48) samples collected from red deer (Cervus elaphus) from north-eastern Croatia in the 2002--05 hunting season were analysed for cadmium (Cd), copper (Cu), iron (Fe), mercury (Hg), lead (Pb), selenium (Se) and zinc (Zn) using atomic absorption spectrometry. Statistical evaluation of results showed age-related accumulations of renal cortex Cd and Zn, bone Pb, and muscle Zn. Renal cortex Cd and Zn were significantly associated. In addition, concentrations of Cd and Pb in muscle tissue were significantly correlated with Fe content. Found levels of toxic metals were not likely to affect the health status of animals. A total of 49% of the muscle, 60% of the kidney and 6% of the liver samples were unsuitable for human consumption according to Croatian regulations for Cd in food. However, the calculated intake of Cd through deer meat consumption is small and represents no health risk when consumption is moderate.

A matrix effect in pectin-rich fruits hampers digestion of allergen by pepsin in vivo and in vitro.

BACKGROUND: It is a general belief that a food allergen should be stable to gastric digestion. Various acidic plant polysaccharides, including pectin, are ubiquitous in fruit matrixes and can form hydrogels under low-pH conditions. OBJECTIVE: The purpose of this study was to investigate the effect of hydrogel forming polysaccharide-rich fruit matrixes on in vivo gastric and in vitro pepsic digestion of fruit allergens. METHODS: Fruit extract proteins (kiwi, banana, apple and cherry) and a purified major kiwi allergen Act c 2 were digested with simulated gastric fluid in accordance with the US Pharmacopeia. In vivo experiments on kiwi fruit digestion were performed on four healthy non-atopic volunteers by examining the gastric content 1 h after ingestion of kiwi fruit. The Act c 2 and kiwi proteins were detected in immunoblots using monoclonal anti-Act c 2 antibodies and rabbit polyclonal antisera. RESULTS: Crude fruit extracts were resistant to digestion by pepsin when compared with commonly prepared extracts. In the gastric content of all volunteers, following kiwi fruit ingestion and immunoblotting, intact Act c 2 was detected with anti-Act c 2 monoclonal antibodies, while kiwi proteins of higher molecular weights were detected using rabbit polyclonal antisera. Addition of apple fruit pectin (1.5% and 3%) to the purified kiwi allergen was able to protect it from pepsin digestion in vitro. CONCLUSION: The matrix effect in pectin-rich fruits can influence the digestibility of food proteins and thereby the process of allergic sensitization in atopic individuals.

Mercury, arsenic, lead and cadmium in fish and shellfish from the Adriatic Sea.

The aim was to measure concentrations of total mercury, total arsenic, lead and cadmium in common edible fresh fish and shellfish from various areas of the Adriatic Sea. Estimates of intake of these elements were made through seafood consumption by the general population. Samples were either wet digested for mercury and arsenic, or dry ashed for lead and cadmium analysis. Mercury was measured by cold vapour atomic absorption spectrometry (CV AAS) and arsenic, lead and cadmium by electrothermal atomic absorption spectrometry (ET AAS). Quality control procedures of analytical methods, which included analyses of dogfish muscle-certified reference material DORM-2, confirmed the acceptability of methods. The highest mercury and arsenic concentrations were found in hake (Merluccius merluccius) and the lowest in mackerel (Scomber scombrus). The respective values in hake were 0.373 +/- 0.075 and 23.3 +/- 3.6, and in mackerel 0.153 +/- 0.028 and 1.06 +/- 0.29 mg x kg(-1) fresh weight (mean +/- SD). Lead and cadmium concentrations were about 10 times higher in shellfish than in analysed fish. The highest lead and cadmium concentrations were found in mussel (Mytilus galloprovincialis) and the lowest in hake. Respective lead and cadmium values in mussel were 0.150 +/- 0.009 and 0.142 +/- 0.017, and in hake were 0.007 +/- 0.004 and 0.002 +/- 0.001 mg x kg(-1) fresh weight. The concentrations of analysed elements were below acceptable levels for human consumption set by the Croatian Ministry of Health, except for total arsenic. The estimated intake of those trace elements included in this study through seafood consumption by the general population did not exceed the provisional tolerable weekly intake recommended by the Joint FAO/WHO Expert Committee on Food Additives.

Placental cadmium and progesterone concentrations in cigarette smokers.

Cadmium and progesterone concentrations were evaluated in term placentas collected from 56 healthy parturients in the city of Zagreb. Concentrations of lead, iron, zinc, and copper in placentas were analyzed. Data collected by questionnaire identified 29 nonsmoking and 27 smoking women. From each placenta, three samples from different locations were taken. Metals were measured by atomic absorption spectrometry. Progesterone was determined by specific radioimmunoassay in homogenized and lyophilized tissue samples after steroid extraction with ethanol. No effect of sample location was found. In placentas of smoking women an increase in cadmium, reduced progesterone and a decrease in iron concentrations were found. Placental copper and zinc concentrations were not altered. In conclusion, the results present new evidence that maternal smoking reduces placental progesterone content and support the established association of smoking with placental cadmium.

Succimer treatment during ongoing lead exposure reduces tissue lead in suckling rats.

There is a concern that oral treatment with succimer (meso-2, 3-dimercaptosuccinic acid, DMSA) can promote gastrointestinal lead absorption if not performed in a lead-safe environment. The scope of our investigation was to evaluate the efficacy of oral DMSA treatment during oral lead exposure on tissue lead in suckling rats. Six-day-old Wistar rats of both genders were divided into two groups-untreated (Pb) and treated (Pb + DMSA)-with 10 animals per group. Lead (as acetate) was given orally at a dose of 2 mg kg(-1) body weight day(-1) for eight consecutive days (total dose 16 mg kg(-1), i.e. 0.08 mmol kg(-1)). During this period the treated group received a daily dose of 0.5 mmol DMSA kg(-1) body weight p.o. six times on days 1-3 and 6-8 of the experiment (total dose 3 mmol kg(-1)). Tissue lead was determined by means of atomic absorption spectrometry. The DMSA efficiently reduced the lead concentration in the analysed tissues (carcass, liver, kidneys and brain) by approximately 50% compared with untreated controls. The pups' growth and organ weights were not affected. In conclusion, our results indicate that DMSA is an efficient oral lead chelator in sucklings even if challenged with ongoing lead exposure.

Elimination of mercury from amalgam in rats.

The aim of this study was to measure the urinary mercury excretion in rats exposed to amalgam over a two months period. Animals were either exposed to mercury from 4 dental amalgams or fed the diet containing powdered amalgams. The results showed significantly higher mercury amount in urine of both exposed groups than in control. Even two months after the amalgam had been placed in rats teeth, the amount of mercury in the urine remained 4-5 times higher than in control, and 4 times higher than in rats exposed to diet containing powdered amalgam. The elevated urinary Hg amount was accompanied by an increased level of total protein in urine. In the same exposure period the excretion of total protein in urine of rats with amalgam fillings was 2 times higher than in control and 1.5 times higher than in rats exposed to amalgam through diet. Concentrations of mercury in the sera of all groups were below the detection limit of the method. The results show that amount of mercury and protein in the urine of rats were related to the mercury release from dental malgam.

Lead, cadmium, and mercury dietary intake in Croatia.

This paper gives an overview of published data on levels of lead, cadmium, total and methyl-mercury in various food items, and of a daily dietary intake assessment in Croatia focusing on the last 10 years. It briefly describes the most reliable methods for quantitative analysis of lead, cadmium, and mercury in biological material. Lead and cadmium concentrations in vegetables and in organs of domestic animals refer to non-exposed rural areas. Mercury concentrations in fish and mussels refer to industrially polluted and non-polluted areas of the Adriatic. The daily dietary intake of lead and cadmium was assessed on the basis of duplicate-diet-collection and food-disappearance method. The assessment of the total and methyl-mercury dietary intake was based on dietary surveys of family seafood consumption. Lead and cadmium intake through food in the general population was 6-40% of the Provisional Tolerable Weekly Intake (PTWI), depending on the assessment method. The only Croatian population consuming more mercury through seafood is the one living in Dalmatia, approaching the PTWI defined by WHO.

Calcium supplementation efficiently reduces lead absorption in suckling rats.

The effect of calcium supplementation on tissue lead was evaluated in suckling Wistar rats. Such data are not yet available in the literature. The following artificial feeding regimen was used for calcium supplementation: cow's milk by addition of 1%, 3% or 6% Ca as CaHPO(4)x2H(2)O suspension to increase the daily calcium intake about 1.4, 2 or 3 times above control values. Artificial feeding was applied during 7 hr each day for nine consecutive days (from day 6 through 15 after birth). The effect of such treatment on lead absorption and elimination was evaluated in two separate experiments: calcium supplementation during oral lead exposure (as acetate; daily dose 2 mg Pb/kg body wt.; total Pb dose 18 mg/kg body wt.) or after a single intraperitoneal lead administration (5 mg/kg body wt.). At the end of experiments, lead in tissues (liver, kidneys, brain and carcass), and essential elements (Ca, Fe, Zn, Cu) were analysed by atomic absorption spectrometry. Calcium supplementation caused a statistically significant decrease of lead in all tissues of sucklings orally exposed to lead. This decrease was dose-related being about 1.3, 1.5 and 2 times lower in groups supplemented with 1%, 3%, or 6% calcium compared to controls, respectively. Increased calcium intake had no effect on incorporated lead after parenteral lead exposure. Calcium supplementation increased carcass calcium and had no effect on trace elements in tissues, pups' general appearance and body weight gain. It is concluded that higher calcium intake might be a way of efficient reduction of lead absorption during the suckling period.

Uptake of cadmium, copper, iron, manganese, and zinc in mushrooms (Boletaceae) from Croatian forest soil.

The concentration of trace elements (Cd, Cu, Fe, Mn, and Zn) was measured in different species of mushrooms (Boletaceae) and correlated with corresponding elements in soil. Five different species of Boletaceae mushrooms and soil samples were collected from forests of Varazdin county in Croatia. Trace elements were analyzed by atomic absorption spectrometry in mushrooms and in EDTA-extracted soil. The results showed that Cd, Cu, and Zn are concentrated in mushroom tissue from soil with transfer factors (mushroom/soil) of 27.0,10.5, and 12.5, respectively. Cadmium incorporated much less in Leccinum (mean 0.73 mg/kg dry weight) than in Boletus, Xerocomus, or Gyroporus (respective means, 6.8, 8.4, and 12.3 mg/kg). Copper and Zn were accumulated in all collected mushrooms (14.7-35.6 and 109-179 mg/kg, respectively) with no difference among species. There was no accumulation of Fe and Mn in mushrooms, but concentrations differed between species, with lowest values in Leccinum. Iron varied from 31 to 878 mg/kg and Mn from 2.9 to 409 mg/kg. Correlations between elements in mushrooms and soil were significant only for Mn. Because no significant correlations for Cd, Cu, Zn, and Fe between mushrooms and soil were found, more studies are needed with only one species of mushrooms collected at locations with different levels of soil contamination. In spite of higher concentrations of Cd in some Boletaceae species, it is assumed that Cd intake through moderate mushroom consumption remains below suggested Provisional Tolerable Weekly Intake (FAO/WHO).

Mobilisation of cadmium by meso- and racemic-2,3-dimercaptosuccinic acid (DMSA) in rats.

A higher efficiency of cadmium binding with racemic than with meso-2,3-dimercaptosuccinic acid (rac-DMSA; meso-DMSA) was found in an in vitro speciation model by Fang et al. (1996). This finding has not yet been tested in vivo. This paper presents results on mobilisation of cadmium by meso- and rac-DMSA in rats. Cadmium chloride was administered as the radioactive isotope 109Cd intraperitoneally to all animals. One group was an untreated control and two groups were treated with meso- and rac-DMSA, respectively. Treatment with chelators was applied twice, immediately after 109Cd and 24 hr afterwards intraperitoneally at the dose of 1 mmol/kg, each. Six days later radioactivity was measured in the liver and kidneys. Whole-body counting was carried out on days 1, 2, 3 and 6 of the experiment. At the end of the experiment, both treatments caused a decrease in 109Cd whole-body retention with rac-DMSA being more efficient (decrease from 83% in control to 74% and 64% in groups treated with meso- and rac-DMSA, respectively). The same reduction of 109Cd was obtained by both chelators in the liver (from 57% to about 47%). In the kidney only rac-DMSA produced significant reduction of 109Cd (from 5.3% to 3.5%). In conclusion, these results show modest reduction of cadmium in the body by two isoforms of DMSA with rac-DMSA being slightly more efficient than meso-DMSA.

Chelation of aluminium by combining DFO and L1 in rats.

The hypothesis that two known chelators 1, 2-dimethyl-3-hydroxypyrid-4-one (L1) and desferrioxamine (DFO) might be more efficient as combined treatment than as monotherapies in removing aluminium from the body was tested in a new acute rat model. Five-week old female rats received chelators: L1 (p.o.), DFO (i.p.) or L1+DFO as 100 or 200 mg/kg dose half an hour after a single i.p. administration of 6 mg Al/kg body weight in the form of chloride. Serum aluminium concentration and urinary aluminium and iron excretions were determined by electrothermal or flame atomic absorption spectrometry. Both chelators were effective only at the higher dose level. While DFO was more effective than L1 in enhancing urinary aluminium excretion, L1 was more effective than DFO in enhancing urinary iron excretion. In the combined treatment group L1 did not increase the DFO effect on aluminium and DFO did not increase the effect of L1 on iron elimination. However, in this group a simultaneous increase in both aluminium and iron elimination was observed. Our results support the usefulness of this animal model for preliminary in vivo testing of aluminium chelators. Urinary values were more useful because of the high variability of serum results. Result of combined chelators treatment should be confirmed in a different experimental model before extrapolation to other systems. This testing procedure of course does not provide all the relevant answers for evaluating the efficiency of chelating agents in aluminium toxicity.

Cadmium inhibits vacuolar H(+)ATPase-mediated acidification in the rat epididymis.

In rats, an acidic luminal pH maintains sperm quiescence during storage in the epididymis. We recently showed that vacuolar H(+)ATPase-rich cells in the epididymis and vas deferens are involved in the acidification of these segments. Treatment of rats with cadmium (Cd) leads to alkalinization of this fluid by an unknown mechanism. Because Cd may affect H(+)ATPase function, we examined 1) the in vivo effect of Cd poisoning on H(+)ATPase-rich cell morphology and on the abundance and distribution of the 31-kDa H(+)ATPase subunit in cells along the rat epididymis, and 2) the in vitro effect of Cd on H(+)ATPase activity and function in the isolated vas deferens. Immunofluorescence and immunoblotting data from rats treated with Cd for 14-15 days (2 mg Cd/kg body mass/day) showed that 1) H(+)ATPase-positive cells regressed to a prepubertal phenotype, and 2) H(+)ATPase was lost from the apical pole of the cell and was redistributed into an intracellular compartment. In experiments in vitro, Cd inhibited bafilomycin-sensitive ATPase activity in isolated total cell membranes and, as measured using a proton-selective extracellular microelectrode, inhibited proton secretion in isolated vas deferens. We conclude that alkalinization of the tubule fluid in the epididymis and vas deferens of Cd-treated rats may result from the loss of functional H(+)ATPase enzyme in the cell apical domain as well as from a direct inhibition of H(+)ATPase function by Cd.

Dental amalgam mercury exposure in rats.

The aim of this study was to measure the distribution of mercury, in tissues of rats exposed to amalgam over a two months period. Possible interaction of mercury with copper and zinc in organs was also evaluated. Rats were either exposed to mercury from 4 dental amalgams, or fed the diet containing powdered amalgam during two months. Mercury was measured in the kidney, liver and brain, copper in kidney and brain and zinc in kidney. The results showed significantly higher concentrations of mercury in the kidneys and the brains of rats in both exposed groups compared to control. Even after two months of exposure to mercury brain mercury concentration in rats with amalgam fillings was 8 times higher than in the control and 2 times higher than in rats exposed to amalgam supplemented diet. The highest mercury concentration in the latter group was found in the kidneys and it was 5 times higher than in the control group. We found no significant differences between mercury levels in exposed and control rat's liver. Exposure to mercury from dental amalgams did not alter the concentrations of copper and zinc in the tissues. Histopathological analyses of rats tissues did not show any pathological changes. These results support previously proposed nose-brain transport of mercury released from dental amalgam fillings.

Combined chelation therapy in reducing tissue lead concentrations in suckling rats.

The very young are more prone to lead poisoning than adults, and the treatment with chelating agents, either as monotherapy or combined treatment, is still a matter of dispute. The purpose of this work was to evaluate the efficiency of three chelating agents administered either as monotherapies or as combined treatments in sucklings. Lead acetate (5 mg Pb kg(-1) i.p.) was administered to the 7-day-old rat pups in eight litters on experimental day 1 and chelating agents on experimental days 2 and 3. Pups were divided into six groups: (1) untreated control; (2) EDTA (calcium disodium ethylendiaminetetraacetate, 0.3 mmol kg(-1) i.p. at 4 p.m.); (3) meso-DMSA (meso-2,3-dimeracaptosuccinic acid, 0.5 mmol kg(-1) p.o. at 10 a.m.); (4) rac-DMSA (racemic-2,3-dimeracaptosuccinic acid, 0.5 mmol kg(-1) p.o. at 10 a.m.); (5) EDTA+meso-DMSA; and (6) EDTA+rac-DMSA. Rats were killed on experimental day 5. Tissue element concentrations were analyzed by atomic absorption spectrometry. Treatment with EDTA did not affect tissue Pb, but it reduced Zn in the carcass and liver. Meso-DMSA reduced Pb in the kidneys and brain, and it did not affect organ essential elements. Rac-DMSA most efficiently reduced Pb concentrations in the carcass, kidneys and brain, but it also reduced Zn and Cu in the liver and Zn in the kidneys. Combined treatments with EDTA never improved the efficiency of either DMSA isoform in decreasing tissue Pb but they did reduce tissue Zn concentrations. All treatments caused the same decrease in the carcass Ca concentrations. The results do not support combined treatment in this age group, which is especially sensitive to trace element deficiencies, and suggest that meso-DMSA might be the treatment of choice in acute lead poisoning in infants.

Comparison of two methods using atomic absorption spectrometry for determination of selenium in food.

This study describes and compares two methods of atomic absorption spectrometry (AAS) for the selenium (Se) analysis in food: electrothermal AAS (ET AAS) and hydride generation method of AAS (HG AAS). The accuracy of the two methods was established by analysing two biological reference materials: Wheat Flour 1567a and Bovine Liver 1577b from the National Institute of Standards and Technology, USA. Good agreement with certified values was obtained for both methods. The accuracy of ET AAS method is 109% and 103% for Wheat Flour and Bovine Liver, respectively. The respective accuracies for HG AAS method were 88% and 87%. The detection limit obtained for ET AAS was 1 microgram Se/L and for HG AAS 0.02 microgram Se/L. The repeatability of ET AAS method was 5-11% and that of HG AAS 14-17%. Both methods are similar in accuracy and repeatability, but hydride generation (HG AAS) is more sensitive than graphite furnace technique (ET AAS) for determination of selenium in food.

Meso-2,3-dimercaptosuccinic acid in the treatment of occupationally exposed lead workers.

The aim of this study was to evaluate the efficacy of meso-2,3-dimercaptosuccinic acid (DMSA) treatment in workers with increased lead absorption and no overt symptoms of lead poisoning. Seven occupationally lead exposed male workers with blood lead concentrations (PbB) exceeding 50 micrograms/100 ml and a positive calcium disodium ethylenediaminetetraacetate (EDTA) lead mobilization test were treated with DMSA for 19 days. Individual doses were 700 mg DMSA, three times a day from day one to five, and twice a day from day six to 19. The treatment intensified urinary lead excretion, most rapidly during the first five days. The increased elimination was followed by a decline of mean PbB to 15% of the pretreatment values. However, 15 days after the treatment, the PbB concentrations rebounded, yet kept below the baseline values and did not exceed 40 micrograms/100 ml. After repeated EDTA lead mobilization test, urine lead was 23-68% of that before DMSA treatment. It can be concluded that DMSA can effectively reduce chelatable lead in occupationally exposed workers.

Comparison of two methods for destruction of biological material for determination of selenium.

The purpose of the study was to compare two methods for destruction of biological material for selenium (Se) analysis: wet digestion by conductive heating in programmed digestion block and digestion in microwave oven. In both methods samples were prepared in a closed system using nitric acid. Selenium was analysed by electrothermal atomic absorption spectrometry. The results have shown that both methods are convenient for complete mineralisation and are accurate in determining selenium in a variety of foodstuffs. Microwave digestion, however, has the advantage of speed and simplicity over the conventional heating procedure.

Comparison of two extraction procedures for determination of trace metals in soil by atomic absorption spectrometry.

The study compares nitric acid and ethylenediaminetetraacetic acid (EDTA) extraction of forest soils for the analysis of trace metals Fe, Mn, Zn, Cu, Pb, and Cd. Fifty forest soil samples from ten different locations were extracted using both methods. The two extraction procedures were compared through regression analysis for each element. All elements were analysed by flame atomic absorption spectrometry method (AAS). The results show that both extracting procedures are only partially effective for most of the measured trace elements in soil. The extraction with diluted HNO3 was incomplete, as large amounts of siliceous material remained undigested. Recoveries obtained by this method for Fe, Mn, Zn, Cu, Pb, and Cd on certified reference material (San Joaquin Soil) were 34, 79, 47, 56, 71, and 102%, respectively. EDTA extraction was even less effective and is considered to reflect the quantity of bioavailable metals. The respective recoveries were 2, 45, 7, 20, 38 and 74%. The regression analyses performed for EDTA-extractable vs. HNO3-extractable metals of forest soils showed high and significant correlation for all examined metals, except Fe.

Validation of microwave digestion method for determination of trace metals in mushrooms.

A microwave digestion method for mushrooms, developed in the study, allows fast preparation of samples and reduces the contamination risk in the process of determining trace metals. Concentrations of six trace elements, Fe, Mn, Cu, Zn, Pb, and Cd were measured in 50 samples of different species of edible mushrooms (fam. Boletaceae) using atomic absorption spectrometry after microwave and dry ashing procedure. The methods were validated through certified Standard Reference Material SRM 1577b (Bovine Liver) which was treated and analysed using the same procedures as for the mushrooms. The samples were either digested with concentrated HNO3 in closed Teflon PFA vessels in a microwave oven, or ashed in quartz crucibles at 450 degrees C. The respective recoveries of Fe, Mn, Zn, Cu, Pb, and Cd obtained by measuring SRM were 112, 107, 104, 115, 111 and 95% after microwave digestion procedure and 86, 101, 109, 111, 98, and 110% after dry ashing procedure. The correlation between concentrations obtained by the two different methods of sample preparation was high for all metals.