Per Leffler

Mono-(2-ethylhexyl) phthalate stimulates basal steroidogenesis by a cAMP-independent mechanism in mouse gonadal cells of both sexes

Phthalates are widely used as plasticizers in a number of daily-life products. In this study, we investigated the influence of mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the frequently used plasticizer di-(2-ethylhexyl) phthalate (DEHP), on gonadal steroidogenesis in vitro. MEHP (25-100 microM) stimulated basal steroid synthesis in a concentration-dependent manner in immortalized mouse Leydig tumor cells (MLTC-1). The stimulatory effect was also detected in KK-1 granulosa tumor cells. MEHP exposure did not influence cAMP or StAR protein levels and induced a gene expression profile of key steroidogenic proteins different from the one induced by human chorionic gonadotropin (hCG). Simultaneous treatment with MEHP and a p450scc inhibitor (aminoglutethimide) indicated that MEHP exerts its main stimulatory effect prior to pregnenolone formation. MEHP (10-100 microM) up-regulated hormone-sensitive lipase and 3-hydroxy-3-methylglutaryl coenzyme A reductase, suggesting that MEHP increases the amount of cholesterol available for steroidogenesis. Our data suggest that MEHP, besides its known inhibitory effect on hCG action, can directly stimulate gonadal steroidogenesis in both sexes through a cAMP- and StAR-independent mechanism. The anti-steroidogenic effect of DEHP has been proposed to cause developmental disorders such as hypospadias and cryptorchidism, whereas a stimulation of steroid synthesis may prematurely initiate the onset of puberty and theoretically affect the hypothalamic-pituitary-gonadal axis.

Impact of water saturation level on arsenic and metal mobility in the Fe-amended soil

The impact of water saturation level (oxidizing-reducing environment) on As and metal solubility in chromium, copper, arsenic (CCA)-contaminated soil amended with Fe-containing materials was studied. The soil was mixed with 0.1 and 1 wt% of iron grit (Fe(0)) and 1, 7 and 15 wt% of oxygen scarfing granulate (OSG, a by-product of steel processing). Solubility of As and metals was evaluated by a batch leaching test and analysis of soil pore water. Soil saturation with water greatly increased As solubility in the untreated as well as in the Fe-amended soil. This was related to the reductive dissolution of Fe oxides and increased concentration of As(III) species. Fe amendments showed As reducing capacity under both oxic and anoxic conditions. The cytotoxicity of the soil pore water correlated with the concentration of As(III). The Fe-treatments as well as water saturation of soil were less significant for the solubility of Cu, Cr and Zn than for As. The batch leaching test used for waste characterization substantially underestimated As solubility that could occur under water-saturated (anaerobic) conditions. In the case of soil landfilling, other techniques than Fe-stabilization of As containing soil should be considered.

Cadmium-metallothionein nephrotoxicity in the rat: Transient calcuria and proteinuria

After a s.c. injection of 0.4 mg Cd/kg as cadmium-metallothionein (CdMT) in rats, a marked increase in urinary protein concentration appeared at 16-40 h. There was a peak of urinary Cd content during the first 4 h after the treatment. Urinary Ca was increased at 8 h after the CdMT injection and returned to normal level at 32 h. Luminal and basolateral renal membrane vesicles were isolated from both control group and CdMT (0.4 mg Cd/kg) group at 24 h after the injection. Calcium uptake and binding of both fractions were decreased in the group treated with CdMT. Cd, Zn and MT concentrations in the kidney cortex were increased, but Ca concentration was not significantly changed. Since injected CdMT is probably only partly reabsorbed by tubular cells at the dose level of 0.4 mg Cd/kg as CdMT, excessive plasma CdMT is rapidly excreted in urine, explaining the increased Cd excretion during the first few hours observed in the present experiment. Decreased Ca binding in the luminal membranes as observed in vitro could be one of the mechanisms of production of calcuria if occurring in vivo. Another possible explanation of calcuria is that Cd ions released from CdMT into the cytoplasm of the tubular cell, may exert ionic interference with Ca transport across the luminal membranes and produce decreased Ca reabsorption. It is known that a disturbance of Ca metabolism could influence the membrane stability and such a change may contribute to explaining the proteinuria characteristic of CdMT nephrotoxicity. The reversibility of the proteinuria observed after a single dose of CdMT may be related to the induction of metallothionein synthesis in the renal cells.

The susceptibility of spontaneously diabetic mice cadmium-metallothionein nephrotoxicity

Cadmium metallothionein (CdMT) was injected subcutaneously into obese hyperglycaemic Umeå ob/ob mice or their lean litter mates (normal mice) at doses of 0, 0.1 and 0.4 mg Cd/kg. Proteinuria and calciuria were induced in both types of mice, but in the ob/ob mice this condition developed at a lower dose of CdMT (0.1 mg Cd/kg) than in the normal mice (0.4 mg Cd/kg). These results show, therefore, that Umeå ob/ob mice are particularly susceptible to CdMT-induced nephrotoxicity. The mechanism underlying this phenomenon needs to be further investigated. After the administration of CdMT, a dose-related increase in glycosuria was observed in both types of mice, in spite of decreased levels of serum insulin and glucose. It is suggested that such glycosuria induced by CdMT could be one of the signs of cadmium nephrotoxicity. The results of the present study thus indicate that metabolic changes like those in diabetes may increase susceptibility to cadmium-induced renal tubular damage.

Differential calcium transport disturbances in renal membrane vesicles after cadmium–metallothionein injection in rats

Cadmium-metallothionein (CdMT) induced calciuria may result from disturbed calcium (Ca) transport through the renal tubular epithelium. The present study aimed at defining time of onset and the degree of disturbed calcium transport. Kidneys were obtained from rats at 4, 12 and 24 h after a single injection of CdMT (dose 0.4 mg Cd/kg b.w.), and compared to saline injected controls. Rapid-filtration 45Ca-assays were performed on basolateral and luminal membrane vesicles, isolated from kidney cortex using a sequential ultracentrifugation procedure. Luminal 45Ca uptake was increased at 4 h and then declined to about 80% of controls, suggesting an early phase perturbation of Ca absorption. Basolateral 45Ca uptake was reduced to less than 50% of controls, starting already at 4 h while 45Ca binding was reduced at 8 h. This may reflect an inhibited basolateral Ca pump mechanism after the binding step. Since the Ca pump normally expels Ca from the cell, an accumulation of intracellular calcium was indicated. Metal analysis verified a four-fold increase of Ca in kidney cortex at 24 h. This suggests that Cd impact on tubular cells involves disturbances on cellular absorption as well as expulsion of Ca.

Techniques for the Stabilization and Assessment of Treated Copper-, Chromium-, and Arsenic-contaminated Soil

Abstract Remediation mainly based on excavation and burial of the contaminated soil is impractical with regard to the large numbers of sites identified as being in need of remediation. Therefore, alternative methods are needed for brownfield remediation. This study was conducted to assess a chemical stabilization procedure of CCA-contaminated soil using iron (Fe)-containing blaster sand (BS) or oxygen-scarfing granulate (OSG). The stabilization technique was assessed with regard to the feasibility of mixing ameliorants at an industrial scale and the efficiency of the stabilization under different redox conditions. The stability was investigated under natural conditions in 1-m3 lysimeters in a field experiment, and the effect of redox conditions was assessed in a laboratory experiment (10 L). The treatments with high additions of ameliorant (8% and 17%) were more successful in both the laboratory and field experiments, even though there was enough Fe on a stochiometric basis even at the lowest addition rates (0.1% and 1%). The particle size of the Fe and the mixing influenced the stabilization efficiency. The development of anaerobic conditions, simulated by water saturation, increases the fraction of arsenic (AsIII) and, consequently, As mobility. The use of high concentrations of OSG under aerobic conditions increased the concentrations of nickel (Ni) and copper (Cu) in the pore water. However, under anaerobic conditions, it decreased the As leaching compared with the untreated soil, and Ni and Cu leaching was not critical. The final destination of the treated soil should govern the amendment choice, that is, an OSG concentration of approximately 10% may be suitable if the soil is to be landfilled under anaerobic conditions. Alternatively, the soil mixed with 1% BS could be kept under aerobic conditions in a landfill cover or in situ at a brownfield site. In addition, the treatment with BS appeared to produce better effects in the long term than treatment with OSG.

Interactions Between pH, Potassium, Calcium, Bromide, and Phenol and Their Effects on the Bioluminescence of Vibrio fischeri

Little attention has been paid to how the light produced by the bacterium Vibrio fischeri in the Microtox assay is dependent on the concentration of essential ions such as sodium and potassium, and whether the concentrations of these ions affect the sensitivity of the test system to toxic chemicals. Five selected factors, pH, potassium (K+), calcium (Ca2+), bromide (Br−), and phenol (Phe), were simultaneously varied over a set of systematically planned experiments according to a D-optimal design that supported the estimation of a model with linear, quadratic, and two-factor interatcions of the studied factors. The bacterial light production represented by the gamma values in the Microtox assay for the 24 selected combinations of factors was measured at 5 and 15 min. The gamma values varied from negative to positive values greater than 1, indicating stimulation and inhibition of bacterial light production, respectively. The relationship between the gamma values and the factor settings was investigated with multiple linear regression. After 5 min of exposure, the light production was significantly affected by linear and quadratic terms for K+, pH, and Phe and an interaction between pH and Phe. The situation was more complex after 15 min of exposure, since in addition significant interactions were found for K × Phe and Ca × pH. The tolerance of V. fischeri to Phe was enhanced by increasing the K and Ca concentrations. Data indicate that the ion composition and pH of the sample, as well as the diluents, need to be considered when the toxicity of salts, water samples, and extracts of sediments and soils are tested using commercially certified toxicity test kits.

Environmental Hazard Screening of a Metal-polluted Site Using Pressurized Liquid Extraction and Two In Vitro Bioassays

Abstract Rapid screening methods can improve the cost effectiveness, throughput, and quality of risk assessments of contaminated sites. In the present case study, the objective was to evaluate a combination of pressurized liquid extraction and 2 in vitro bioassays for the hazard assessment of surface soil sampled from 46 points across a pyrotechnical industrial site. Pressurized liquid extraction was used to rapidly produce soil-water extracts compatible with 2 high-capacity bioassays. Hazard assessment using combined toxicological and chemical screening revealed zones with relatively high potential risks of metal pollution. Multivariate data analysis provided indications that significant inhibition in the bioassays was correlated with levels of metals in the extracts, suggesting an elevated toxic potential from certain metals. Low pH and high concentrations of dissolved organic carbon were associated with increased cytotoxicity of extracts, indicating that these factors influence metal bioavailability. The cytotoxicity observed was more strongly correlated to metal concentrations in the extracts than in the soil, suggesting that measurements of total metal concentrations in soils do not provide good indications of the soils potential toxicity.

Cadmium-metallothionein-induced kidney dysfunction increases magnesium excretion in the rat

Urinary excretion of the major minerals, calcium (Ca), magnesium (Mg), sodium (Na), and potassium (K), as well as of protein and metallothionein, was studied following the injection of cadmium-metallothionein (CdMT) in rats. Animals were given vehicle (saline) and 0.4 and 0.8 mg Cd/kg body wt as CdMT. A marked, relatively early, and reversible increase in Mg excretion was seen. The increase was dose-related, indicating a close connection to the typical Cd-derived cellular damage in the renal tubular epithelium, including an early reversible Ca excretion and a late reversible protein excretion. The increase in Mg excretion was similar in magnitude to the one for Ca and much more prominent than that recorded for Na and K. The appearance of Mg and Ca excretion peaks at an early stage after CdMT injection makes it likely that this effect is an early event in the process of development of cellular damage and does not merely represent unspecific cellular damage giving rise to proteinuria.

Toxicity and accumulation of trinitrotoluene (TNT) and its metabolites in Atlantic salmon alevins exposed to an industrially polluted water.

A pond in an industrial area in Sweden was selected to study adverse effects on salmon alevins from 2,4,6-trinitrotoluene (TNT)-contaminated water. Chemical screening revealed heavy contamination of TNT and its degradation products, 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT), ranging from 0.05 to 230 g/kg in the sediment (dry weight) within the water system. Pond water contained 3 mg/L TNT. A dilution series of pond water mixed with tap water revealed increased death frequency in alevins down to fivefold dilution (approximate 0.4 mg TNT/L). Uptake was concentration dependent, reaching 7, 9, and 22 μg/g tissue for TNT, 2-ADNT, and 4-ADNT at the highest test concentration. A time-dependent uptake of TNT and its degradation products was found at a water concentration of 0.08 mg TNT/L. Degradation products of TNT showed a more efficient uptake compared to native TNT, and accumulation of 4-ADNT was more pronounced during the late phase of the 40-d exposure study. Bioconcentration factors (BCF) (0.34, 52, and 134 ml/g for TNT, 2-ADNT, and 4-ADNT, respectively) demonstrated a significant uptake of the metabolite 4-ADNT in alevin tissue. Disturbed physiological conditions and delayed development in alevins were not studied, but may not be excluded even at 125-fold diluted pond water (0.016 mg TNT/L). BCF data indicated that bioaccumulation of TNT metabolites need to be considered in TNT chronic toxicity. Fish species and age differences in the accumulation of TNT metabolites need to be further studied.