Cadmium transport by Chlorella pyrenoidosa

Chlorella pyrenoidosa, incubated at 31°C in a medium containing CdCl₂ concentrations ranging from 0.025 to 0.500 μg/ml, incorporated cadmium in a linear fashion for 60 min. The rate of uptake was dependent upon the exogenous cadmium concentration; 2.98 μM CdCl₂ was required to half-saturate the transport system. Cd uptake is reduced by lower temperatures and when cultures are not illuminated. Both manganese and iron inhibited cadmium uptake but zinc and cobalt had no effect. The inhibition of cadmium uptake by manganese could be correlated with the concentration of manganese present; kinetic studies showed manganese to be a competitive inhibitor. Similar kinetic studies revealed that Mn transport is competitively inhibited by cadmium. The ability of C. pyrenoidosa to incorporate cadmium and manganese is determined, in large part, by the composition of the growth medium. Cells, grown in a medium containing low levels of Mn, incorporate both Cd and Mn at a faster rate than cells grown in a medium containing high levels of Mn. The rate of Cd and Mn incorporation is also faster in cells that have been previously exposed to Cd. The data support the conclusion that manganese and cadmium share a common transport system in C. pyrenoidosa.     

Cadmium uptake by the crayfish, Orconectes propinquus propinquus (Girard)

Orconectes propinquus was exposed to 10, 100, and 1000 ppb Cd(Cl₂) containing 0.09 μCi/liter ¹⁰⁹ Cd(Cl₂) for 1.5, 4.5, 10.5, 22.5, 46.5, 94.5, and 190.5 hours. At 10 ppb, total Cd uptakes between 1.5 and 94.5 hours were not significantly different. By 190.5 hours, the organisms had accumulated a mean concentration of 18.4 ppm Cd, which was significantly higher than the concentrations accumulated at earlier times. At 100 ppb, Cd uptake at 1.5 hours was significantly less (P < 0.05) than that at 22.5–190.5 hours and uptake at 4.5 hours was significantly less than that at 94.5 and 190.5 hours. Also, uptake at 10.5 hours was significantly less than that at 190.5 hours. Uptakes were not significantly different between 22.5 and 94.5 hours; but were significantly higher than at 1.5 hours and lower than at 190.5 hours. At 1000 ppb, uptake increased with time and was significantly greater (P < 0.05) at every time interval monitored. By 190.5 hours, the organisms had accumulated a mean Cd concentration of 534.4 ppm. At all time intervals at 1000 ppb, Cd uptake was significantly higher (P < 0.01) than that at 100 and 10 ppb. Uptakes at 100 and 10 ppb were not significantly different.     

The effect of beryllium on the growth of human lung fibroblasts

The effect of beryllium on the proliferative capacity of human fetal lung fibroblasts (IMR-90) was investigated. The addition of BeCl₂ to cultures, at concentrations ranging from 0.02 to 0.50 μg Be/ml, increased the population doubling time and decreased the maximal growth level in a dose-dependent manner. Upon achieving maximal growth, cultures exposed to beryllium concentrations of 0.05 μg/ml and above began to decline in cell number, presumably because of cell death. Studies with ⁷BeCl₂ showed that fibroblasts began to remove beryllium from the growth medium within 24 hr after dosing and continued to accumulate beryllium for 120 hr; after that time, the amount of beryllium per cell appeared to level off. Cells grown in 0.05 μg Be/ml accumulated 2.5 times more beryllium than cells grown in 0.02 μg Be/ml. Exposure of fibroblasts to a concentration of 0.005 μg Be/ml had no detectable effect on the number of population doublings after a single subculture, but reduced the population doublings by 42% after the second subculture. Comparative studies using different beryllium compounds showed that beryllium citrate had a greater inhibitory effect than BeCl₂, which was, in turn, more inhibitory than BeSO₄; BeO, even at a concentration of 2.0 μg Be/ml, had no effect. The degree to which fibroblast growth is inhibited by a particular beryllium compound may be related to the ease with which the cell can accumulate the beryllium complex present.     

A comparison between fecal cadmium and urinary beta2-microglobulin, total protein, and cadmium among Japanese farmers. An epidemiological study of cooperation between Japan and Sweden.

A study was made of 156 farmers living in a cadmium-exposed area and 93 farmers in a reference area. All were between 50 and 69 years of age. Cadmium intake from food was estimated from daily fecal cadmium content, body burden from urinary cadmium concentration, and cadmium-induced renal effects from urinary β₂-microglobulin (β₂-m) and total protein concentration. Average cadmium intake in the reference area was about 40 μg/day and in the exposed area about 150 μg/day. Average urinary cadmium excretion in the reference group was 2 μg/liter and in the exposed group 7.5 μg/liter. Average urinary β₂-m concentration in the reference group was 86 μg/liter and we defined tubular proteinuria as a β₂-m concentration higher than the average plus two standard deviations. With this definition the prevalence rate of tubular proteinuria was 3% in the reference group and 14% in the exposed group. Tubular proteinuria increased with age and with exposure duration. Increased total proteinuria was also more common in the exposed group but the prevalence rate ratio was 2.4 as compared to 4.4 for tubular proteinuria.     

Effect of pH on cadmium toxicity,speciation, and accumulation during naphthalene biodegradation

Lowering pH of a microbiological medium from 7 to 4 decreased cadmium toxicity during naphthalene biodegradation by a Burkholderia sp. Cadmium speciation and cadmium accumulation in the system were studied to explain this effect. Cadmium speciation was determined by direct measurement and by geochemical modeling. Previous studies have implicated the monovalent hydroxylated cadmium (CdOH+) species in the effect of pH on cadmium toxicity. Modeling analysis predicted CdOH+ formation only at very low concentrations (< or = 0.0128 microM), while the measured concentration of divalent ionic cadmium (Cd2+) was at least three orders of magnitude greater, suggesting that Cd2+ is the more significant metal form. With respect to cadmium accumulation, cells contained in media adjusted to pH 4 accumulated only 2.76 +/- 0.76 mg Cd/g cells, whereas cells in media adjusted to pH 7 accumulated 8.52 +/- 0.71 mg Cd/g cells. These data suggest that cadmium toxicity is correlated with increased cadmium accumulation rather than the formation of CdOH as pH is increased. At low pH, the decrease in cadmium accumulation may be caused by increased competition between hydrogen and cadmium ions for binding sites on the cell surface or by an increase in metal efflux pump activity due to an increase in the proton gradient that drives the efflux pump.     

The effects of cadmium-accumulated chlorella on the reproduction of Moina macrocopa (cladocera)

Chlorella cultured for 6 days in a medium containing cadmium at 0.02, 0.1, and 0.5 ppm accumulated approximately 14, 70, and 340 μg/g (in dry wt) of cadmium, respectively. These chlorella were supplied as food to Moina macrocopa everyday after washing with artificial soft water. Reproductive impairment was not observed in M. macrocopa fed chlorella containing 14 μg/g of cadmium. Slight reproductive impairment was observed in M. macrocopa fed chlorella containing 70 gmg/g of cadmium at initial and late stages of the experiment. However, survival rate, growth rate, and reproductive rate were significantly affected in M. macrocopa fed chlorella containing 340 μg/g of cadmium. The accumulation level of cadmium in chlorella which causes 50% reproductive impairment of M. macrocopa was estimated to be 280 μg/g (in dry wt). The cadmium accumulation level in M. macrocopa fed these chlorella was measured throughout the 8-day experiment. The rate of accumulation of cadmium by Moina through chlorella was considerably lower than that through the water containing dissolved cadmium.     

Transfer of cadmium in a phytoplankton-oyster-mouse food chain

The trophic transfer of cadmium (Cd) was studied in a phytoplankton-oyster-mouse food chain. Phytoplankton, grown in a continuous culture chemostat system containing CdCl₂ plus the isotope¹⁰⁹Cd, accumulated 70% of the total supplied cadmium. Oysters filtered out 85 to 95% of the phytoplankton. The rate of oyster Cd accumulation at 15 C increased linearly with seawater Cd concentration according to: y=0.066X – 0.15 (n =12, r=0.96); where X=g Cd/L seawater (between 2 and 22) and y=g Cd/g dry wt oyster/ day. About 59% of the Cd accumulated by the oysters came from the phytoplankton food source and 41% from the cadmium dissolved in the water. Sixty-one percent of the total supplied cadmium was retained in the whole soft body of the osyters. Mice fed 0.4 g of oyster-bound Cd per g of diet, retained 0.83% of the dietary cadmium consumed. Mouse kidney retention for organic oyster-bound cadmium was 0.14%. Extrapolation of these results to human risk assessment indicates that moderate consumption of oysters, which are not highly contaminated with cadmium, poses no significant health risk in terms of elevating kidney cadmium levels.     

Silicon Influence on Maize, Zea mays L., Hybrids Exposed to Cadmium Treatment

Thirty Zea mays L. hybrids were screened using hydroponically—grown seedlings treated in the medium with high cadmium content (100 μM Cd(NO₃)₂·4H₂O). Measurements showed conspicuous differences between the hybrids in the growth parameters in Cd treated plants. Hybrids differed greatly in Cd accumulation and translocation. Root/shoot ratio in Cd concentration ranged from 2.78 to 12.83. The majority of the heavy metal was localized in the root system. Five hybrids were chosen and the effect of silicon (5 mM) effect on high-level cadmium toxicity symptoms was investigated. Silicon decreased Cd accumulation in roots and its translocation into the shoots.     

Effects of Heavy-Metal Stress on Cyanobacterium <Emphasis Type="Italic">Anabaena flos-aquae</Emphasis>

The influence of two metals, copper and cadmium, was studied on the growth and ultrastructures of cyanobacterium Anabaena flos-aquae grown at three different temperatures: 10°C, 20°C, and 30°C. The highest concentration of chlorophyll a was observed at 20°C and the lowest at 10°C. Both toxic metal ions, Cu²⁺ and Cd²⁺, inhibited growth of the tested cyanobacterium. Chlorophyll a concentration decreased with the increase of metal concentration. A 50% decrease in the growth of A. flos-aquae population, compared with the control, was reached at 0.61 mg l^–1 cadmium and at 0.35 mg l^–1 copper (at 20°C). Copper at all temperatures tested was proven to be more toxic than cadmium. At 3 mg l^–1, the lysis and distortion of cells was observed; however, after incubation at 9 mg l^–1 cadmium, most of the cells were still intact, and only intrathylakoidal spaces started to appear. Copper caused considerably greater changes in the protein system of A. flos-aquae than did cadmium; in this case, not only phycobilins but also total proteins were destructed. The aim of this study was also to identify the place of metal accumulation and sorption in the tested cyanobacterium. Analysis of the energy-dispersion spectra of the characteristic x-ray radiation of trichomes and their sheaths showed that cadmium was completely accumulated in cells but was not found in the sheath. Spectrum of the isolated sheath after treatment with copper exhibited only traces of the metal, but isolated cells without a sheath showed a high peak of copper.     

Toxicity and bioaccumulation of cadmium in the colonial green algaScenedesmus obliquus

A laboratory investigation was conducted to study the extent and efficiency of cadmium bioaccumulation inScenedesmus obliquus by subjecting this alga to varied sublethal Cd concentrations. The influence of cell population age on Cd bioaccumulation was also studied. Under the experimental conditions employed, growth was not significantly affected by Cd concentrations ranging from 0.01 ppm to 1.00 ppm. At concentrations above 1.00 ppm, however, growth was inhibited markedly. Increases in external Cd concentration caused an increase in total bioaccumulation over the entire range of concentrations, which did not significantly affect growth. Efficiency of Cd bioaccumulation was also concentration dependent, but maximum accumulation efficiency occurred in a medium with a Cd concentration lower than that medium in which maximum total bioaccumulation occurred. Age of the cell population influenced the extent of Cd bioaccumulation. Rapidly growing, young cultures accumulated less Cd than older cultures approaching stationary growth phase.     

Dose-response analysis of cadmium-induced tubular proteinuria: a study of urinary beta2-microglobulin excretion among workers in a battery factory.

The study covered 240 male and female workers exposed to cadmium oxide and nickel hydroxide dust in a Swedish battery factory. The control group comprised 87 unexposed males. Air samples from the plant showed the present exposure level to be about 50 μg Cd/m³ air. Cadmium-induced effects were studied by measuring urinary β₂-microglobulin excretion. Urinary β₂-microglobulin concentration followed a log-normal distribution in the reference group with a geometric mean of 84 μg/liter (adjusted to a specific gravity of 1.023). In the group of 185 persons continuously exposed to cadmium dust in the work environment, the prevalence of increased urinary β₂-microglobulin excretion increased with employment time. The prevalence was 19% for the workers with 6–12 years of exposure to about 50 μg Cd/m₃ as compared to 3% for those in the reference group. Smokers had about three times higher prevalence than nonsmokers.     

Effects of hexavalent chromium in rainbow trout (Salmo gairdneri) after prolonged exposure at two different pH levels

Toxic effects were studied in rainbow trout (Salmo gairdneri) exposed to Na₂CrO₄ solutions of different concentrations (0.02, 0.2, and 2.0 mg/liter Cr) and pH (7.8 and 6.5). The study involved embryo-through-juvenile and alevin-through-juvenile exposure for 32 weeks and exposure of yearling trout for 1, 3, 6, and 12 weeks. The experiments were started at the same time of the year and were carried out concurrently. When survival was used as the criterion, Cr was more toxic at pH 6.5 than at pH 7.8 in all life stages studied. For trout in the embryo-through-juvenile exposure study the lowest concentrations inducing an increase in mortality were 0.2 mg/liter Cr at pH 6.5 and 2.0 mg/liter Cr at pH 7.8. Embryo hatchability was only affected at the highest exposure concentration at a pH of 6.5. No growth retardation was detected at the termination of the experiments. In addition to survival and growth, biochemical, hematological, and histological changes were studied as indicators of toxicity in yearling trout. These parameters also showed that fish were more susceptible to Cr at the lower pH. The observed effects are discussed in relation to previously reported findings in short-term toxicity tests.     

Aroclor 1016: Toxicity to and uptake by estuarine animals

Bioassays were conducted to determine the acute toxicities of the polychlorinated biphenyl (PCB) Aroclor 1016 in flowing sea water to American oysters (Crassostrea virginica), brown shrimp (Penaeus aztecus), grass shrimp (Palaemonetes pugio), and pinfish (Lagodon rhomboides), and to determine its chronic toxicity to, and uptake and retention by pinfish. Acute 96-hour EC50's or LC50's were: oysters, 10.2 μ/liter; brown shrimp, 10.5 μg/liter; grass shrimp, 12.5 μg/liter. The PCB was not toxic to pinfish at 100 μg/liter for 96 hours, but significant mortality occurred when pinfish were exposed to 32 μg/liter of Aroclor 1016 for 42 days. Pinfish exposed to 1 μg/liter for 56 days accumulated the chemical with maximum concentrations attained in whole-fish by 21 to 28 days. Maximum whole-body residue (wet weight) was 17,000 × the nominal concentration in test water. Tissue alterations, such as severe vacuolation in the pancreatic exocrine tissue surrounding the portal veins, occurred in pinfish exposed to 32 μg/liter of Aroclor 1016 for 42 days.     

Maintenance of infectivity of Trypanosoma congolense in vitro with explants of infected skin at 37 degrees C

Glossina morsitans infected with two stocks of Trypanosoma congolense were fed on rabbits and calves to produce local skin reactions containing trypanosomes. Areas of infected skin were removed from the animals and used to prepare dermal explant cultures in Eagle's MEM and RPMI 1640 culture medium, supplemented with foetal bovine serum and containing penicillin and streptomycin. Cultures were incubated at 37 °C and media were changed at 24 to 48 hour intervals to maintain pH 7·0 to 7·2. There was evidence of trypanosome multiplication in explant cultures set up in both media; one trypanosome stock was maintained equally well in both Eagle's MEM and RPMI 1640, but the other stock survived better in Eagle's MEM. Explant cultures prepared from calf tissues generally yielded more trypanosomes at 24 hours than those prepared from rabbit tissues. The numbers of parasites present near the explants at 24 hours were maintained for up to 14 to 15 days before a decline in parasite concentration occurred. The organisms retained typical blood stream trypomastigote morphology and were infective for mice for periods up to 21 days. The trypanosomes growing in primary explant cultures could not be subpassaged in culture media alone or on to monolayers of fibroblast-like cells of bovine, murine or buffalo origin. Attempts to establish primary cultures by placing infected skin explants directly on to similar monolayers were also unsuccessful.     

Cadmium IC50 determinations on Chlorella vulgaris involving different parameters

The ecotoxicity of cadmium nitrate has been tested, at the basic trophic level, on a chlorophycea alga, Chlorella vulgaris. Several parameters of growth, under strictly defined conditions, have been selected: opacimetry at 665 nm, cell count biomass volume estimation (Coulter counter), and ATP and chlorophyll levels. Dose-response curves have been established and correspond to the percentage of inhibition of culture growth as a function of Cd²⁺ concentration in the medium up to 3 mg/liter. The 50% inhibition of culture growth is defined as the Cd²⁺ concentration (IC₅₀) inducing a 50% reduction of the number of cells compared to the control. So, it is clearly pointed out that the size of the inoculum is of great importance. The IC₅₀ determinations by cell count give lower values than by other methods: on the contrary, opacimetry gives higher values. These differences are not surprising with regard to the meanings of the various criteria. Therefore, both the selected parameters and the calculation method can greatly modify the IC₅₀ values. Anyway, for routine purposes, opacimetry is a rapid and sufficiently satisfactory screening procedure, but other parameters have to be taken in account in second steps.     

Urinary beta2-microglobulin excretion among people exposed to cadmium in the general environment: an epidemiological study in cooperation between Japan and Sweden.

In a cadmium-exposed area, 138 farming women between 51 and 60 years of age and 40 reference women in the same age group were studied. Cadmium exposure was estimated from data on cadmium concentration in the individual farm's rice production, and on individual residence times. Possible cadmium exposure from the consumption of river water was also taken into account. The average urinary cadmium concentration was about twice as high in the exposed group as in the reference group. Cadmium levels in the blood among the exposed persons were also considerably elevated. Urinary β₂-microglobulin excretion was measured quantitatively as an indicator of renal tubular changes. Increased excretion was strongly related to residence time in the exposed area as well as to the use of contaminated river water in the household. Among the 35 women who had lived only at one place within the exposed area and who had not consumed river water, there was a tendency toward increased β₂-microglobulin excretion with an increased cadmium level in rice and increased residence time. There was also a correlation between cadmium levels in the blood and β₂-microglobulin excretion.     

Toxicity and accumulation of selenite and selenate in the unicellular marine alga Cricosphaera elongata

The toxicity and bioaccumulation of selenium in the 4⁺ and 6⁺ oxidation states were investigated in a marine unicellular alga Cricosphaera elongata in culture. Selenite was more toxic than selenate. Exponentially growing cells and cells in the stationary phase of C. elongata rapidly accumulated selenite (0.1 and 1.0 mg/L Na₂SeO₃) and selenate (0.1 and 1.0 mg/L Na₂SeO₄). Within the first two hours of contact, the amount of selenium taken up decreased sharply in exponentially growing cells, while cells in the stationary phase continued accumulating selenium until a plateau was reached. The presence of metabolic inhibitors such as KCN (potassium cyanide) or DCMU (3-(3,4-dichloro-phenyl)-1,1 dimethylurea) or glutaraldehyde did not modify the first phase of accumulation of selenite by C. elongata in the stationary phase, whereas further accumulation was inhibited. Possible mechanims of accumulation of selenium are discussed. In a series of long term experiments (14 or 31 d), intracellular partitioning of Se in C. elongata cells, exposed to selenite, was analysed; total, protein-bound and free cytosolic selenium concentrations increased with selenium concentration added to the culture medium (0.1 or 1 mg/L Na₂SeO₃) and with exposure time (at 0.1 mg/L Na₂SeO₃) from 14 d or 31 d. Most of the selenium was associated with proteins; these proteins may represent a form of storage or detoxication of selenium.     

Accumulation and Elimination of Trace Metals in a Transplantation Experiment with Crassostrea rhizophorae

Oysters, Crassostrea rhizophorae, were reciprocally transplanted to two different sites: a contaminated site in the Cotegipe Channel at Aratu Bay and an uncontaminated site at Cacha-Prego, inside and outside of Todos os Santos Bay (Brazil), respectively. Trace metal accumulation was measured after 0, 15, 30, and 60 days of exposure at the contaminated site. Oysters transplanted for 60 days from the clean to the contaminated site had accumulated cadmium and lead to similar concentrations as found in the native oysters. They had also accumulated copper and zinc, but to lower levels compared to native specimens. Elimination experiments were carried out by transplanting oysters in the reverse direction. After 30 days, concentrations of cadmium and lead had decreased to levels comparable to those in the native specimen, whereas concentrations of copper and zinc did not diminish. A second elimination experiment, bringing back to Cacha-Prego oysters that had been exposed 60 days at Cotegipe Channel, indicated stronger decreases of copper and lead, but no clear changes of cadmium and zinc concentrations. The accumulation experiment with C. rhizophorae is useful to estimate trace metal bioavailability and changes in concentrations as a function of time at the contaminated site. The different results of elimination experiments in the uncontaminated site suggest different degrees of trace metal fixation after long-term and short-term accumulation periods. Received: 1 July 1998/Accepted: 12 May 1999     

Investigation of Chlorella pyrenoidosa Protein as a Source of Novel Angiotensin I-Converting Enzyme (ACE) and Dipeptidyl Peptidase-IV (DPP-IV) Inhibitory Peptides

Chlorella pyrenoidosa (C. pyrenoidosa) is a microalgae species with a remarkably high protein content that may potentially become a source of hypotensive and hypoglycemic peptides. In this study, C. pyrenoidosa proteins were extracted and hydrolyzed overnight with pepsin and trypsin with final degrees of hydrolysis of 18.7% and 35.5%, respectively. By LC-MS/MS, 47 valid peptides were identified in the peptic hydrolysate (CP) and 66 in the tryptic one (CT). At the concentration of 1.0 mg/mL, CP and CT hydrolysates inhibit in vitro the angiotensin-converting enzyme (ACE) activity by 84.2 ± 0.37% and 78.6 ± 1.7%, respectively, whereas, tested at cellular level at the concentration of 5.0 mg/mL, they reduce the ACE activity by 61.5 ± 7.7% and 69.9 ± 0.8%, respectively. At the concentration of 5.0 mg/mL, they decrease in vitro the DPP-IV activity by 63.7% and 69.6% and in Caco-2 cells by 38.4% and 42.5%, respectively. Short peptides (≤10 amino acids) were selected for investigating the potential interaction with ACE and DPP-IV by using molecular modeling approaches and four peptides were predicted to block both enzymes. Finally, the stability of these peptides was investigated against gastrointestinal digestion.     

Effects of bisulfite on metabolic development in synchronous Chlorella pyrenoidosa

Addition of bisulfite to synchronous cultures of Chlorella pyrenoidosa adversely affects both active growth and cell propagation. Analyses of chlorophyll, protein, RNA, and DNA revealed decreases of all these components with the decline in cell population, as HSO₃^? concentration was increased. Biochemical analysis after treatment showed a differential change in the levels of chlorophylls, protein, RNA, and DNA, when expressed as percentages of the cell dry weight: the content of chlorophyll b increased, whereas that of chlorophyll a decreased gradually with increases in HSO₃^?. The amounts of protein and RNA per unit dry weight remained constant, but DNA levels were markedly reduced. With the exception of iron, the levels of nutrient elements within the cells increased with increasing HSO₃^? concentration. The relative increases were most marked with K, Mg, Ca, Cu, and Na. The results suggest that HSO₃^? adversely affects cell development by impairing DNA synthesis.