Effect of polyaspartic acid on CdCl2-induced nephrotoxicity in the rat

We produced an animal model of CdCl₂ nephrotoxicity in rats, and treated them with polyaspartic acid (PAA) to prevent renal damage. Male Sprague-Dawley (SD) rats (190–200 g) were used to induce proximal renal tubular damage by daily injection of CdCl₂ 3.0 mg/1,000 g body wt for 2 wk. CdCl₂-exposed SD rats exhibited significant increases in urine volume, urinary excretion ofN-acetyl-β-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), and fractional excretion of sodium (FENa) and a decrease in the percentage of tubular reabsorption of phosphate (%TRP). Of these indicators of proximal tubular function, AAP and %TRP are more sensitive than NAG or FENa. No glycosuria or aminoaciduria, however, were observed. PAA markedly improved these indicators of proximal tubular function. Daily urinary protein excretion and creatinine clearance, on the other hand, did not change after administration of PAA. Cd concentrations in the cortex were 3 times higher than in the medulla, however, there were no differences between Cd-treated rats and PAA-treated rats. Our animal model is an excellent one for determining the effect of cadmium on renal proximal tubule damage. PAA appears to be useful in the treatment of CdCl₂ nephrotoxicity.     

No meaningful increase in urinary tubular dysfunction markers in a population with 3μg cadmium/g creatinine in urine

The critical Cd exposure level to induce tubular dysfunctions is a focus of public concern among general populations in Japan. To answer this question, one group each (about 1000 adult women/area) in nonpolluted areas with high (Area H) and low Cd exposure (Area L) was obtained, and 742 strictly age-matched pairs of never-smoking adult women were selected for comparison. Cd, α₁-MG (microglobulin) and β₂-MG in urine were taken as markers of exposure and tubular dysfunction, respectively. Geometric mean Cd levels as corrected for creatinine (Cd_cr) was greater than three times higher in Area H (2.8 μg/g cr) than in Area L (0.8 μg/g cr). Nevertheless, β₂-MG_cr did not differ between the two areas (125 μg/g cr for Area H vs 118 μg/g cr for Area L). α₁-MG_cr was only marginally higher in Area H (2.8 mg/g cr) than in Area L (2.1 mg/g cr), with no biomedical significance. Results were essentially the same when analyses were conducted with noncorrected observed values or values corrected for a specific gravity. Thus, the effects of Cd exposure in Area H on renal tubular function should be essentially nil.     

Low Cadmium Levels in Urine of Residents in two Prefectures where Cadmium Levels in Locally Harvested Brown Rice are Higher than in other Prefectures in Japan

Recent publications in Japan suggest that Cd in river beds and locally harvested rice tend to be higher in seven prefectures in the north-eastern part on the coast of the Sea of Japan (the high-Cd zone). The present study was initiated to investigate the current level of exposure to Cd and possible health effects among local populations in the zone. Thus, levels of Cd and three tubular dysfunction markers [i.e., α ₁-MG, β ₂-MG, and N-acetyl-β-d-glucosaminidase (NAG)] were examined in urine of local residents (700 and 704 adult women, respectively) in two prefectures (prefecture 1 and prefecture 7), which were located in the north-east (prefecture 1) and south-west ends (prefecture 7) of the high-Cd zone. Urinary Cd levels [e.g., 0.99 and 0.78 μg/l as geometric mean (GM) for observed (non-corrected) values, respectively] in prefecture 1 and prefecture 7 were comparable to the levels in other parts of Japan (All Japan-A; 0.99 μg/l). Correspondingly, GM values for α ₁-MG (2.29 and 1.99 mg/l vs. 2.17 mg/l for All Japan-A) and for β ₂-MG (87 and 80 μg/l vs. 99 μg/l for All Japan-A) were not elevated, and NAG also stayed unchanged (2.89 and 2.87 units/l for prefecture 1 and prefecture 7, respectively). Evaluation in combination with the findings in other five prefectures in the zone suggests that Cd exposure is equal to the national average both in prefecture 1 and in prefecture 7, whereas Cd exposure appeared to be elevated in the central part of the zone. The observation appears to be on line with geographical location of the two prefectures that they are on the two ends of the zone of high natural Cd background.     

Mortality in a cadmium polluted area in Japan

A 15-year follow-up study of 3178 inhabitants (1424 men and 1754 women) living in the cadmium (Cd) polluted Kakehashi River basin was conducted. The results clarified effects on mortality of renal dysfunction induced by Cd indicated by urinary beta-2-microglobulin (beta2-MG), total protein, glucose, and total amino acids. This study used Cox's proportional hazard model. The mortality risk ratio of urinary beta2-MG positive (>= 1000 microg/gCr) subjects was significantly increased in both sexes: 1.35 for men and 1.73 for women. The increased mortality ratio of the urinary protein positive (>= 10 mg/dl) subjects was also significant for both sexes, with risk ratios of 1.82 for men and 2.01 for women. Only the women showed significantly increased mortality of the urinary glucose positive (>= 20 mg/dl) subjects and amino acids positive (> = 300 microg/gCr) subjects. When the subjects were divided into four categories according to urinary beta2-MG, <300, 300-1000, 1000-10000, >= 10000 microg/gCr, the mortality risk ratios were increased in proportion to the increase of urinary beta2-MG in both sexes. These results suggest that mortality of Cd-exposed subjects increased with increasing excretion of four urinary markers of renal tubular dysfunction, and in proportion to increases in the amount of urinary beta2-MG excretion including under 1000 microg/gCr.     

Urinary Scandium as Predictor of Exposure: Effects of Scandium Chloride Hexahydrate on Renal Function in Rats

Some of the rare earth elements such as Sc are believed to be non-toxic and, at present, are widely utilized for the replacement of toxic heavy metals in technological applications, but they are not entirely free of toxicity, with hidden potential health risks. In this animal experiment, we report the urinary scandium (Sc) excretion rate and nephrotoxiciy in male Wistar rats. For this purpose, the rats were given a single dose of a solution of scandium chloride by intraperitoneal injection. The Sc excretion (U-Sc) was determined in 24-h urine samples by inductively coupled plasma–argon emission spectrometry along with the Sc nephrotoxicity, urine volume (UV), creatinine (Crt), β-2-microglobulin (β2-MG) and N-acetyl-β-d-glucosaminidase (NAG). A dose-dependent Sc excretion of 0.0063% (r = 0.97) via 24-h urine was confirmed. The administration of Sc induced a significant decrease of UV and Crt and a significant increase of NAG and β2-MG. These results suggest that U-Sc can be a useful tool for monitoring Sc exposure. The formation of Sc colloidal conjugates that deposit in glomeruli may be the cause of a reduction of the glomerular filtration rate. We propose that the analytical method and results described in this study will be of great importance for future toxicological studies on Sc exposure.     

Urinary Profile of 6β-Hydroxycortisol in Workers Exposed to Aluminum

The aim of the present study was to evaluate whether aluminum exposure may cause alterations of cytochrome P ₄₅₀3A4 by measuring the urinary excretion of 6β-hydroxycortisol as biomarker. There is a positive correlation between urinary 6β-hydroxycortisol and aluminum excretion (p < 0.05). The correlations between occupational duration, smoking habits, and age were also evaluated. The increased excretion of the biomarker suggests that this element may induce formation of cytochrome P ₄₅₀ in exposed individuals.     

Estimation of the reference level of urinary cadmium by using hybrid method in cadmium-polluted and non-polluted areas in China

This study investigated the reference level of urinary cadmium (UCd) of two Cd-polluted areas and one non-polluted area in China by applying the updated hybrid approach. Urinary N-acetyl-β-d-glucosaminidase (NAG) and β-2-microglobulin (β₂MG) were considered as indicators of early renal dysfunction. UCd was regarded as an indicator of the total internal Cd exposure. We recruited 3285 inhabitants for this investigation. Among them, 2106 were from polluted areas, while the other 1179 were from non-polluted areas. BMDL₁₀ (the lower 95% confidence limit of the benchmark dose (BMD)) of UCd for NAG was 1.55 μg/g creatine in men and 1.59 μg/g creatine in women. Using β₂MG as the renal effect marker, BMDL₁₀ of UCd was 1.58 μg/g and 1.53 μg/g in men and women, respectively. Our BMDL estimates accounting for age were lower than those reported in previous studies, suggesting that policies to control Cd exposure are warranted.     

Detoxification of cadmium in tobacco plants: formation and active excretion of crystals containing cadmium and calcium through trichomes

In tobacco (Nicotiana tabacum L.), long and short trichomes can be distinguished morphologically. The established function of long trichomes is to exude a sticky gum containing diterpenes, whereas that of short trichomes is not known. When tobacco seedlings were exposed to toxic levels of cadmium (Cd), growth was retarded, but trichome number was increased up to 2-fold in comparison with untreated samples. Observation by variable-pressure scanning electron microscopy (VP-SEM) indicated that large crystals of 150 μm in size were formed on head cells of both short and long trichomes. An energy-dispersive X-ray analysis system fitted with VP-SEM revealed the crystals to contain amounts of Cd and calcium (Ca) at much higher concentrations than in the head cells themselves. Transmission electron microscopy demonstrated crystal formation in amorphous osmiophilic deposits in vacuoles. When seedlings were treated with Cd in the presence of Ca, tolerance was increased in proportion to the increase in Ca concentration. These results indicate that tobacco plants actively exclude toxic Cd by forming and excreting Cd/Ca-containing crystals through the head cells of trichomes. Received: 26 June 2000 / Accepted: 20 September 2000     

Genetic and physiological parameters associated with cadmium toxicity in Drosophila melanogaster

Two strains of Drosophila melanogaster represent the extremes in resistance and sensitivity to the lethal effects of CdCl₂. The strain containing the mutations vermilion and brown (v; bw) and the strain Austin had LC₅₀'s of 3.3 and 1.3mm CdCl₂, respectively. The three major chromosomes from these two strains were assorted genetically into the six possible combinations. The measured LC₅₀'s for CdCl₂ for these six genotypes fell into two groups according to the X chromosome; those containing the X chromosome from v; bw had LC₅₀'s 0.5–1.0mm greater than those in which the X chromosome was from Austin. Since the parent strains differed by 2mm, we suggest that the X chromosome is a major, but not the sole, site of genes to produce resistance to CdCl₂. When ¹⁰⁹Cd was in the diet the uptake by v; bw and Austin over 2 days was the same. After 4 days of uptake, the Austin strain excreted the ¹⁰⁹Cd five times faster than v; bw but the six genotypes did not differ appreciably in excretion rate from one another and resembled the sensitive parent Austin more than the resistant one. Thus a second process is indicated that distinguishes resistance to CdCl₂ that apparently is not associated with the X chromosome.This research was sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-84OR21400 with Martin Marietta Energy Systems, Inc.     

Calcium supplementation effect on calcium balance in endurance-trained athletes during prolonged hypokinesia and ambulatory conditions

Calcium (Ca) supplements may be used to normalize Ca-balance changes but little is known about the effect of Ca supplements on Ca balance during hypokinesia (decreased kilometers per day). The aim of this study was to evaluate the effect of daily intakes of Ca supplements on Ca balance during hypokinesia (HK). Studies were done during 30 d of a pre-HK period and during 364 d of a HK period. Forty male athletes aged 23–26 yr were chosen as subjects. They were divided equally into four groups: unsupplemented ambulatory control subjects (UACS), unsupplemented hypokinetic subjects (UHKS), supplemented hypokinetic subjects (SHKS), and supplemented ambulatory control subjects (SACS). The SHKS and UHKS groups were kept under an average running distance of 0.7 km/d. In the SHKS and SACS groups supplemented with 35.0 mg Ca lactate/kg body weight. Fecal Ca loss, urinary excretion of Ca and phosphate (P), serum concentrations of ionized calcium (Ca_I) total Ca, P, and Ca balance, intact parathyroid hormone (iPTH) and 1,25 dihydroxyvitamin D (1,25(OH)2D), anthropometric characteristics and peak oxygen uptake were measured. Fecal Ca excretion, urinary Ca and P excretion, serum Ca_I, total Ca, and P concentration, and negative Ca balanced increased significantly (p ≤ 0.01) in the SHKS and UHKS groups when compared with the SACS and UACS groups. Serum, urinary, and fecal Ca changes were much greater and appeared much faster in the SHKS group than in the UHKS group. Serum iPTH and 1,25 (OH)2 D, body weight, and peak oxygen uptake decreased significantly (p ≤ 0.01) in the SHKS and UHKS groups when compared with the SACS and UACS groups. In contrast, the corresponding parameters remained stable in the SACS and UACS groups when compared with the baseline control values. It was concluded that during prolonged HK, urinary and fecal Ca excretion and serum Ca concentration increased significantly despite the presence of a negative Ca balance; thus, Ca supplements cannot be used to normalize negative Ca balance during prolonged HK.     

Association between urinary calcium excretion level and mortality in inhabitants of the Jinzu River basin area of Japan

A follow-up study on 5442 inhabitants (2699 men, 2743 women) was conducted to determine the association between urinary Ca excretion level and mortality of the general population of three different areas of the Jinzu River basin area, namely, non-Jinzu River, mixed, and the Jinzu River water systems, over a period of 6127 d. More than 98% of the subjects were followed completely in each area. In comparison with the low- and high-Ca-excretion groups (cutoff values; 25.1 mg/dL in men, 20.4 mg/dL in women), the mortality rates per 1000 person-years and standardized mortality ratios (SMRs) tended to be greater in the low-Ca-excretion groups than in the high-Ca-excretion groups in both sexes for each of the three areas. Moreover, Cox’s hazard ratios in men and women of the three areas exhibited negative values, 0.99 except for men of the mixed water system. These values were statistically significant in both sexes for the Jinzu River water system and in women for the non-Jinzu River water system. We conclude that the life-span becomes shorter as urinary Ca excretion levels become lower.     

Degradation of endosulfan during substrate preparation and cultivation of Pleurotus pulmonarius

Summary Endosulfan is an insecticide used on many vegetable crops. In mushroom cultivation, vegetable materials used as a growth substrate may contain residues of endosulfan that may accumulate in the final mushroom biomass. After preparing the substrate, it is subjected to pasteurization and/or composting and then inoculated with the desired fungus. The purpose of this research was to determine the rate and extent of endosulfan reduction from a grass substrate that was either composted or sterilized by autoclaving. In addition, the rate and extent of removal of endosulfan from substrate colonized with Pleurotus pulmonarius was determined. The degradation of 65 mg/kg endosulfan was analyzed on both, the substrate preparation and the culture of P. pulmonarius on the grass Digitaria decumbens. During composting in presence of Ca(OH)₂ for 120 h, the concentrations of α and β endosulfan were reduced by 61.4 and 49.5% respectively, significantly higher compared with the control (without Ca(OH)_2,) in which the reduction was 38.5%. After sterilization the concentration of α and β endosulfan was reduced by 84.8 and 87.5% respectively. After the colonization of substrate by P. pulmonarius (15 days after spawning) α and β endosulfan were reduced by 96% and at the end of cultivation (35 days after spawning) were reduced by 99%. When carpophores were analyzed, residues of α and β endosulfan were observed between 0.019–0.084 mg/kg. The results showed that α and β endosulfan were partially removed during the preparation of substrate and entirely eliminated during fungal colonization on the substrate.     

Phosphate deposition capacity of athletes during hypokinesia, phosphate loading, and ambulation

Hypokinesia (diminished movement) induces significant phosphate (P) excretion; however, little is known about the P deposition ability of the body during hypokinesia (HK). Using P loads, the aim of this study was to establish the deposition ability of the body to retain P during prolonged HK. Studies were done during a 30-d period of pre-HK and a 364-d period of HK. Forty male trained athletes aged 24.7 ± 8.0 yr were chosen as subjects. They were equally divided into four groups: unloaded ambulatory control subjects (UACS), unloaded hypokinetic subjects (UHKS), loaded ambulatory control subjects (LACS), and loaded hypokinetic subjects (LHKS). All hypokinetic subjects were limited to an average walking distance of 0.7 km/d. Loading tests with 85.0 mg of calcium phosphate/kg body weight were performed on the LACS and LHKS. Fecal P loss, urinary calcium (Ca) and P loss, serum P, Ca, and the ionized calcium (Ca_I) levels increased significantly (p≤0.05) in the LHKS and UHKS groups when compared with the LACS and UACS groups, respectively. Serum intact parathyroid hormone (iPTH) and the 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂ D₃] levels decreased significantly (p≤0.05) in the LHKS and UHKS groups when compared with the LACS and UACS groups, respectively. After the P load, significant (p≤0.05) differences were observed between LHKS and UHKS groups regarding serum, urinary, and fecal P changes. Thus, the deposition capacity of P decreased significantly (p≤0.05) more in the LHKS group than in the UHKS group. The deposition of P, fecal P, urinary P and Ca, serum Ca_I, P, Ca, 1,25-(OH)₂ D₃, and iPTH changed insignificantly (p>0.05) in control groups when compared with their baseline values. It was shown that after the P load, significant differences were observed between the loaded and unloaded hypokinetic subjects regarding serum, urinary, and fecal P values and P retention. The oral P load intensified P loss from the body. It was concluded that the higher the P intake increased the greater P loss and the lower P deposition and thus the less likely it is for the P load to benefit hypokinetic subjects.     

The toxicity of cadmium to barley plants as affected by complex formation with humic acid

An ‘alternating solution’ culture method was used to study the effects of chloride ions and humic acid (HA) on the uptake of cadmium by barley plants. The plants were transferred periodically between a nutrient solution and a test solution containing one of four levels of HA (0, 190, 569 or 1710 μg cm⁻³) and one of five levels of Cd (0, 0.5, 1.0, 2.5 or 5.0 μg cm⁻³) in either a 0.006M NaNO₃ or 0.006M NaCl medium. Harvest and analysis of shoots and roots was after nineteen days. The distribution of Cd in the test solutions between Cd²⁺, CdCl⁺ and HA-Cd was determined in a separate experiment by dialysis equilibrium. In the nitrate test solutions Cd uptake was clearly controlled by Cd²⁺ concentration and was therefore reduced by HA complex formation. In the absence of HA, chloride suppressed Cd uptake indicating that Cd²⁺ was the preferred species. However complex formation with Cl⁻ enhanced uptake when HA was present because of an increase in the concentration of inorganic Cd species relative to the nitrate system. The ratio root-Cd/shoot-Cd remained at about 10 across a wide range of shoot-Cd concentrations, from about 3 μg g⁻¹ (sub-toxic) up to 85 μg g⁻¹ (80% yield reduction). The ability of the barley plants to accumulate ‘non-toxic’ Cd in their roots was thus very limited. Humic acid also had no effect on Cd translocation within the plant and the root/shoot weight ratio did not vary with any treatment. At shoot-Cd concentrations in excess of 50 μg g⁻¹, K, Ca, Cu and Zn uptake was reduced, probably the result of root damage rather than a specific ion antagonism. The highest concentration of HA also lowered Fe and Zn uptake and there was a toxic effect with increasing HA concentration at Cd=0. However the lowest HA level, comparable with concentrations found in mineral soil solutions, only reduced yield (in the absence of Cd) by <5% while lowering Cd uptake across the range of Cd concentrations by 66%–25%.     

Change in compactness of inclusion bodies of recombinant β-galactosidase expressed in the <Emphasis Type="Italic">araBAD</Emphasis> promoter system of <Emphasis Type="Italic">Escherichia coli</Emphasis>

We investigated the relevance of the relationship between the compactness of β-galactosidase inclusion bodies (β-gal IBs) and their enhanced enzymatic activity with or without the addition of D-fucose (inducer analog) or methyl α-D-glucopyranoside (α-MG, catabolite repressor) after induction in the araBAD promoter system of Escherichia coli. Experiments conducted to evaluate the solubilization of β-gal IBs in guanidine hydrochloride as well as their trypsin degradation and temperature stability revealed that β-gal IBs expressed in response to the addition of D-fucose or α-MG had a looser structure. Additionally, β-gal IBs expressed when D-fucose or α-MG was added were more quickly solubilized in guanidine hydrochloride or degraded by trypsin-treatment than those produced when these compounds were not added. Moreover, the activity of β-gal IBs expressed when D-fucose or α-MG were added was less stable at various temperatures. Consequently, we deduced that the looser structure of β-gal IBs resulted in enhanced enzymatic activity of β-gal IBs upon addition of D-fucose or α-MG after induction.     

Relationship between tolerance and accumulation characteristics of cadmium in higher plants

Ten plant species belonging to 5 families,i.e., Cruciferae, Cucurbitaceae, Gramineae, Leguminosae, and Solanaceae, were grown in a sand soil at two pH levels. The soil was subjected to an application of CdCl₂ at rates of 0 to 700 mgCd·kg⁻¹ soil. The relationship between Cd concentration in the shoots (tc) and soil (sc; NH₄NO₃ extractable) was expressed by the equation: log (tc)=α+βlog (sc). The coefficients α and β were estimated for each species at each level of soil pH. Plottings of the scores on α and β axes showed that the Cd accumulation characteristics in the plants appeared to depend on the families irrespective of soil pH. Based on theupper critical concentration of Cd in the tops (C_t), the Cruciferae and Leguminosae species were found to be the most and the least tolerant to Cd, respectively. The C_t values correlated exceedingly well with the values of α.     

Effects of ambient temperature and of temperature acclimation on nitrogen excretion and differential catabolism of protein and nonprotein resources in the intertidal snails,Littorina saxatilis (Olivi) and L. obtusata (L.)

Nitrogenous excretion in two snails, Littorina saxatilis (high intertidal) and L. obtusata (low intertidal) was studied in relation to temperature acclimation (at 4° and 21°C), including total N excretion rates, the fraction of urea in N excretion, corresponding O:N ratios and the partitioning of deaminated protein between catabolic and anabolic processes at 4°, 11° and 21°C. Aggregate N excretion rates in both species showed no significant compensatory adjustments following acclimation. Total weight specific N excretion rates at 21°C were higher in standard 3 mg L. saxatilis (739 ng N mg⁻¹ h⁻¹) than standard 5 mg L. obtusata (257 ng N mg⁻¹ h⁻¹) for snails acclimated to 21°C. Comparisons of Q₁₀ values of total weight specific N excretion to Q₁₀ values for weight specific oxygen consumption ({xxV}O₂) between 4° to 11 °C and 11° to 21°C indicated that, while total rates of catabolic metabolism ({xxV}O₂) and protein deamination in L. obtusata were essentially parallel, the relationship between N excretion and {xxV}O₂ in L. saxatilis revealed the partitioning of a larger share of deaminated protein carbon into anabolism at 4° and 21°C than at 11°C. Urea N accounted for a larger share of aggregate N excreted in L. saxatilis than in L. obtusata, but in both species urea N is a greater proportion of total N excreted when acclimated at 4°C (urea N: ammonia N ratio range: 1 to 2.15) than in snails acclimated to 21°C (urea N: ammonia N ratio range: 0.46 to 1.39). Molar O:N ratios indicate that the proportion of metabolism supported by protein catabolism is greater in L. saxatilis (O:N range: 2.5–8.4) than in L. obtusata (O:N range: 7.3–13.0). In both species, regardless of acclimation temperature, the O:N ratios are generally lowest (high protein catabolism) at 4°C and highest at 21°C.     

Bone cadmium and lead in the ancient population from El Hierro, Canary Islands

This study was performed to determine the levels of lead (Pb) and cadmium (Cd) in 63 bone samples of the prehispanic population of the island El Hierro, comparing them with the values obtained on 98 prehispanic samples from Tenerife, Fuerteventura, Gran Canaria, and La Palma, all of them in the Canary Islands, and with eight modern samples who served as controls. Prehispanic individuals from El Hierro showed the lowest bone Pb values of all the archipelago (0.72±1.01 mg/kg), significantly different (F=6.9, p<0.001) from the values obtained for the population of other islands such as Tenerife (4.87±5.36 mg/kg) or Fuerteventura (4.45±7.85 mg/kg) and also from those of the modern population (30.53±14.62 mg/kg). On the other hand, bone Cd, although slightly lower in the ancient population groups, was not significantly different when compared with the modern one. In addition, no differences were observed in bone Cd among the ancient population of the different islands. Bone lead—but not cadmium—kept an inverse significant relationship with the distance of the burial site both to south Spain (r=−0.31) and Atlantic Morocco (r=−0.28, p<0.001 in both cases).     

Potential of active excretion of ammonia in three different haline species of crabs

Isolated perfused gills of stenohaline crabs Cancer pagurus adapted to seawater, brackish water-adapted euryhaline shore crabs Carcinus maenas and freshwater-adapted extremely euryhaline Chinese crabs Eriocheir sinensis were tested for their capacity to excrete ammonia. Gills were perfused with haemolymph-like salines and bathed with salines equal in adaptation osmolality. Applying 100 μmol · l⁻¹ NH₄Cl in the perfusion saline and concentrations of NH₄Cl in the bath that were stepwise increased from 0 to 4000 μmol · l⁻¹ allowed us to measure transbranchial fluxes of ammonia along an outwardly as well as various inwardly directed gradients. The gills of all three crab species were capable – to different extents – of active excretion of ammonia against an inwardly directed gradient. Of the three crab species, the gills of Cancer pagurus revealed the highest capacity for active excretion of ammonia, being able to excrete it from the haemolymph (100 μmol · l⁻¹ NH⁺ ₄) through the gill epithelium against ambient concentrations of up to 800 μmol · l⁻¹, i.e. against an eightfold gradient. Carcinus maenas and E. sinensis were able to actively excrete ammonia against approximately fourfold gradients. Within the three crab species, the gills of E. sinensis exhibited the greatest capacity to resist influx at very high external concentrations of up to 4000 μmol · l⁻¹. We consider the observed capacities for excretion of ammonia against the gradient as ecologically meaningful. These benthic crustaceans protect themselves by burying themselves in the sediment, where, in contrast to the water column, concentrations of ammonia have previously been reported that greatly increase haemolymph levels. Electrophysiological results indicate that the permeabilities of the gill epithelia are a clue to understanding the species-specific differences in active excretion of ammonia. During the invasion of brackish water and freshwater, the permeabilities of the body surfaces greatly decreased. The gills of marine Cancer pagurus exibited the greatest permeability (ca. 250 mS cm⁻²), thus representing practically no influx barrier for ions including NH⁺ ₄. We therefore assume that C. pagurus had to develop the strongest mechanism of active excretion of ammonia to counteract influx. On the other hand, freshwater-adapted E. sinensis exhibited the lowest ion permeability (ca. 4 mS cm⁻²) which may reduce passive NH⁺ ₄ influxes at high ambient levels. Accepted: 14 October 1998     

Effect of black cumin (Nigella sativa) on heart rate, some hematological values, and pancreatic β-cell damage in cadmium-treated rats

This study was designed to investigate the effect of Nigella sativa (NS) on the heart rate, some hematological values, and pancreatic β-cell damage in cadmium (Cd)-treated rats. The rats were randomly grouped into one of three experimental groups: Control, Cd treated, and Cd+NS treated. Each group contained 10 animals. The Cd-treated and Cd+NS-treated groups were injected subcutaneously daily with CdCl₂ dissolved in isotonic NaCl in the amount of 2 mL/kg for 30 d, resulting in a dosage of 0.49 mg Cd/kg/d. The control group was injected with only isotonic NaCl (2 mL/kg/d) throughout the experiment (for 30 d). Three days prior to administration of CdCl₂, the Cd+NS-treated group received the daily intraperitoneal (ip) injection of 2 mL/kg NS until the end of the study; animals in all three groups were fasted for 12 h and blood samples were taken for the determination of the glucose and insulin levels, red blood cell (RBC) and white blood cell (WBC) counts, packet cell volume (PCV), and hemoglobin (Hb) concentration. The heart rates of rats were also measured by a direct writing electrocardiograph before the blood withdrawals. It was found that NS treatment increased the lowered insulin levels, RBC and WBC counts, PCV, and neutrophil percentage in Cd-treated rats. However, the WBC count of Cd-treated rats with NS treatment was still lower than those of control values. NS treatment also decreased the elevated heart rate and glucose concentration of Cd-treated rats. Pancreatic tissues were also harvested from the sacrificed animals for morphological and immunohistochemical examinations. Cd exposure alone caused a degeneration, necrosis, and weak degranulation in the β-cells of the pancreatic islets. In Cd+NS-treated rats, increased staining of insulin and preservation of islet cells were apparent. It is concluded that NS treatment might decrease the Cd-treated disturbances on heart rate, some hematological values, and pancreatic β-cell.