Hepatocarcinogenic potential of di(2-ethylhexyl)phthalate in rodents and its implications on human risk

The plasticizer di(2-ethylhexyl) phthalate (DEHP), to which humans are extensively exposed, was found to be hepatocarcinogenic in rats and mice. DEHP is potentially set free from objects made of synthetic materials (e.g., those used in medicine). Chronically, the greatest amounts are transferred to persons undergoing hemodialysis (up to 3.1 mg/kg b.w. per day) who would thus be considered the individuals most endangered by tumorigenesis. Although toxicokinetics seem to play a certain unclear role in the course of DEHP-related toxicity, toxicodynamic factors appear more decisive. DEHP is a representative of "peroxisome proliferators" (PP), a distinct group of substances that, in rodents, do not only induce peroxisomes but also specific enzymes in other organelles, organ growth, and DNA synthesis. The cluster of the characteristic effects of PP is generally, although perhaps not quite appropriately summarized as "peroxisome proliferation," and is strongest in the liver. The lowest observed effect level (LOEL) and the no observed effect level (NOEL) of peroxisome proliferation in the rat, as determined by the induction of specific enzymes (peroxisomal beta-oxidation, carnitine-acetyl-transferase, cytochrome P-452), DNA synthesis, and hepatomegaly, may be assumed as 50 and 25 mg/kg b.w. per day, respectively. DEHP and other carcinogenic PP are neither genotoxic nor tumor initiators, but they appear to be tumor promoters, also implicating a threshold level for the carcinogenic effect. Although a causal relationship between a particular effect of peroxisome proliferation and hepatocarcinogenesis is as yet unknown, peroxisome proliferation as a whole phenomenon appears to be associated with the potential of tumor induction, as shown by comparison of the relative strength of individual PP and by comparison of species and organ specificities. Likewise, LOEL and NOEL of rodent carcinogenesis, that is, 300 and 50 to 100 mg/kg b.w. per day, respectively, are above but not too far from the corresponding values for the investigated parameters of peroxisome proliferation. Thus, with respect to dose alone, worst-case exposure in hemodialysis patients is at least 16-fold below the LOEL of any characterized PP-specific effect of DEHP and approximately 100-fold below that of DEHP-related tumorigenesis. Also, primates are less responsive to PP than rats with respect to the investigated biochemical and morphological parameters. If this lower primate responsiveness is extrapolated to estimate carcinogenicity in humans, we might thus arrive at an even larger safety margin than when based on exposure alone. Doses of PP hypolipidemics that had clearly induced several indicators of peroxisome proliferation in rats did not cause any clear-cut enhancements in the peroxisomes of patients, even though most of these hypolipidemics were considerably stronger PP than DEHP. Thus, an actual threat to humans by DEHP seems rather unlikely. Accordingly, hepatocarcinogenesis was neither enhanced in workers exposed to DEHP nor in patients treated with hypolipidemics.     

Effects of bis(2-ethylhexyl) phthalate on chromosomes of human leukocytes and human fetal lung cells

Blood from two male and two female donors was exposed at 37degrees for 4 hr to concentrations of 60.0, 6.0, 0.6, and 0.06 mug of a widely used plasticizer, bis (2-ethylhexyl) phthalate, per milliliter of blood. The bis(2-ethylhexyl) phthalate was solubilized with polysorbate 80. Appropriate polysorbate and nonpolysorbate controls also were established. Following the 4 hr of incubation, phytohemagglutinin was added and tissue cultures were established. In addition, human fetal lung cells were exposed in tissue culture to a medium containing 6.0 mug/ml of bis(2-ethylhexyl) phthalate in polysorbate 80 for 5 days. Similar controls also were established for these experiments. Analysis of chromosome preparations from all cultures obtained failed to show any increased evidence of isochromatid and chromatid breaks or gaps or abnormal forms at any studied concentration when compared to the control cultures. In addition, analysis of fetal lung cell preparations for aneuploidy failed to reveal any differences between cells from study and control cultures. This study involved a short-term exposure to bis(2-ethylhexyl) phthalate in various concentrations which did not cause damage in leukocytes or fetal lung cells.     

Studies on the effects of orally administered Di-(2-ethylhexyl) phthalate in the ferret

N,N'2-2,7-Flourenylenebisacetamide (2.7-FAA) was administered orally to 34 female, strain AXC/I rats. Nineteen were pretreated with the extirpation of the salivary glands. The remaining 15 rats received no surgical treatment. Six of these 15 unoperated rats developed mammary carcinomas and seven developed tumors at other sites, whereas none of 19 operated rats had mammary tumors but 8 rats had tumors at other sites. No tumor developed in 4 operated control rats that were on normal diet. The results demonstrate that pretreatment with extirpation of the salivary glands inhibited mammary tumor induction in rats by 2,7-FAA although it did not inhibit induction of other type tumors. The mechanism by which induction of mammary tumors was inhibited is not clear. Ovarian atrophy and hepatic necrosis were observed in both operated and unoperated rats ingesting 2,7-FAA but did not seem to affect carcinogenesis by the carcinogen. Lower body weight was observed in operated rats but it is not considered to be the major cause of a complete inhibition of mammary tumor induction.     

Dysregulation of ornithine decarboxylase activity, apoptosis and Bcl-2 oncoprotein in Syrian hamster embryo cells stage-exposed to di(2-ethylhexyl)phthalate and tetradecanoylphorbol acetate

Perturbations of cell proliferation and death are considered as essential events in the process of carcinogenesis. Thus, two parameters, ornithine decarboxylase (ODC), an enzyme closely related to cell proliferation and transformation, and apoptotic phenomenon are profoundly modified. Using Syrian hamster embryo (SHE) cells, we have examined in the framework of two-stage carcinogenesis (initiation-promotion) the effects of a non-genotoxic [diethylhexylphthalate (DEHP)] or genotoxic [benzo[a]pyrene (BaP)] carcinogen or a non-carcinogenic compound [phthalic anhydride (AP)] on these parameters. Immunoreactive Bcl-2 and Bcl-xL proteins were also investigated following two-stage exposures. Whereas exposures to BaP, DEHP or AP alone did not provoke any modification of ODC activity, the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), strongly increased it. Using two-stage exposure protocol (xenobiotics first, then replacement by TPA-promoter), the ODC activity was higher than that obtained with TPA alone. This superinduction of ODC activity was observed only with the carcinogenic compounds DEHP and BaP. Following the same exposure protocol, spontaneous cellular apoptosis was decreased. Furthermore, Bcl-2 oncoprotein was also upregulated approximately 8- and 11-fold for BaP and DEHP respectively; meanwhile Bcl-xL protein rate did not change. The non-carcinogenic compound AP slightly inhibited spontaneous SHE cell death without ODC superinduction. Exogenous polyamines, putrescine, spermidine and spermine diluted in the medium did not inhibit spontaneous apoptosis. Although inhibition of apoptosis was not specific of carcinogenic compound, both superinduction of ODC activity and inhibition of apoptosis via Bcl-2 upregulation, may cooperate during early stages of the carcinogenic process.     

Oral toxicity of bis(2-ethylhexyl) phthalate during pregnancy and suckling in the Long-Evans rat

Bis(2-ethylhexyl) phthalate (DEHP) is a compound widely used in plastics technology to impart flexibility to rigid polymers. We sought to determine whether the oral exposure of female rats to DEHP during gestation and suckling produces alterations in the litter. Female rats were exposed to different concentrations of DEHP suspended in drinking water (32.5 and 325 microl/litre) from day 1 of pregnancy to day 21 after delivery. Pup body weight gain and kidney, liver and testes weight was measured at different times (21, 28, 35, 42 and 56 days) after birth. Plasma concentrations of DEHP and histopathological alterations in kidneys, liver and testes were also studied. In addition, the ability of female pups (1 month of age) to perform a learned avoidance test, the 'beam walking' test, was evaluated. Perinatal exposure to DEHP produced no statistically significant changes in the body weight gain of offspring. Conversely, it produced a significant decrease in kidney and testes relative weight (organ/body weight) with a significant increase in relative liver weight. Signs of histological damage in kidneys, liver, and particularly testes, were observed. Pups exposed perinatally to the highest concentration of DEHP elicited a significant increase in the time necessary to perform the beam walking test.     

The extraction of iron(III) from aqueous acid solutions by di(2-ethylhexyl)phosphoric acid

The extraction of iron(III) from aqueous solutions containing sulphuric, hydrochloric and nitric acids by di(2-ethylhexyl)phosphoric acid (DEHPA) in kerosene has been investigated under different conditions. As a result, it is found that although extraction is dominated by an ion-exchange reaction, the rate of iron(III) extraction from sulphuric acid solutions to reach equilibrium is relatively slow in comparison with that from hydrochloric or nitric acid solutions. In the extraction from aqueous solutions containing hydrochloric or nitric acid, however, the DEHPA combines with iron(III) according to the solvating reaction at higher aqueous acidity. From studies on the rate of the extraction from sulphuric acid solutions, examined under non-equilibrium, it is confirmed that dependencies of extraction rate on hydrogen ion and DEHPA concentrations are in the first and inverse first orders, respectively. The hydrolyzed species is considered to interpret the extraction mechanism in this system.     

The solvent extraction of lead from chloride solutions using di(2-ethylhexyl)phosphoric acid

Lead has been extracted from chloride solutions into kerosene containing excess di(2-ethylhexyl)phosphoric acid, using various concentrations of metal and extractant. Extraction proceeded readily, but the results indicated that 1.6 hydrogen ions were exchanged per atom of lead extracted, rather than 2.0 as would be expected for a divalent metal ion. Extraction was unaffected by metal concentration over the range studied (0.1–1.0 g l^?1), and the results were highly consistent. The unique log D vs. pH relation allowed simple collection of data for contact stage evaluation by rearrangement of the familiar solvent extraction equations and use of empirically derived constants. An S-shaped extraction isotherm resulted, which limits the ease with which aqueous lead concentrations may be reduced by extraction with di(2-ethylhexyl)phosphoric acid.     

The HMG-CoA reductase inhibitor atorvastatin increases the fractional clearance rate of postprandial triglyceride-rich lipoproteins in miniature pigs

We have previously shown in vivo that the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor atorvastatin decreases hepatic apolipoprotein B (apoB) secretion into plasma. To test the hypothesis that atorvastatin modulates exogenous triglyceride-rich lipoprotein (TRL) metabolism in vivo, an oral fat load (2 g fat/kg body wt) containing retinol (50 000 IU) was given to 6 control miniature pigs and to 6 animals after 28 days of treatment with atorvastatin 3 mg. kg-1. d-1. A multicompartmental model was developed by use of SAAM II and kinetic analysis performed on the plasma retinyl palmitate (RP) data. Peak TRL (d<1.006 g/mL; Sf>20) triglyceride concentrations were decreased 29% by atorvastatin, and the time to achieve this peak was delayed (5.2 versus 2.3 hours; P<0.01). The TRL triglyceride 0- to 12-hour area under the curve was decreased by 24%. In contrast, atorvastatin treatment had no effect on peak TRL RP concentrations, time to peak, or its rate of appearance into plasma; however, the TRL RP 0- to 12-hour area under the curve was decreased by 20%. Analysis of the RP kinetic parameters revealed that the TRL fractional clearance rate was increased significantly, 1.4-fold (3.093 versus 2.276 pools/h; P=0.012), with atorvastatin treatment. The percent conversion of TRL RP from the rapid-turnover to the slow-turnover compartment was decreased by 47% with atorvastatin treatment. The TRL RP fractional clearance rate was negatively correlated with very low density lipoprotein apoB production rate measured in the fasting state (r=-0.49). Thus, although atorvastatin had no effect on intestinal TRL assembly and secretion, plasma TRL clearance was significantly increased, an effect that may relate to a decreased competition for removal processes by hepatic very low density lipoprotein.     

Mass transfer characteristics of cobalt and nickel in di(2-ethylhexyl)Phosphoric acid under steady-state extraction conditions

The mass-transfer characteristics of cobalt and nickel in di(2-ethylhexyl)phosphoric acid have been studied using a gradientless or steady-state stirred cell contactor. The effects of contacting conditions, stirrer Reynolds number, extractant concentration and organic phase loading were investigated. The results showed that under the conditions studied overall transfer rates could be considered diffusion or mass-transfer controlled. There was, however, evidence of an interfacial or chemical resistance as shown by the effect of contacting conditions on values of the overall mass-transfer coefficient, K_org. This was confirmed when experimental K_org values were compared with individual phase mass-transfer coefficient values predicted from correlations proposed in the literature by Lewis and by McManamey et al.     

The extraction of zinc(II) from sulphate and perchlorate solutions by di(2-ethylhexyl)phosphoric acid dissolved in Isopar-H

The extraction of zinc from sulphate and perchlorate solutions at constant ionic strength, by di(2-ethylhexyl)phosphoric acid, DEHPA, in Isopar-H has been studied at 25°C as a function of metal and DEHPA concentrations. The distribution data have been analyzed both graphically and numerically, using the computer program LETAGROPDISTR [33]. The results showed that the species extracted into the organic phase have the composition ZnA₂HA and ZnA₂H₂A₂ for both perchlorate and sulphate systems. The formation constants for each system are given in Table 2. An estimate of the formation constant of ZnSO₄ in aqueous phase was obtained from the extraction data in sulphate solutions.     

Effects of ionizing radiation (neutrons/gamma rays) on plasma lipids and lipoproteins in rats

Two closely related genes, the presenilins ( PS ), located at chromosomes 14q24.3 and 1q42.1, have been identified for autosomal dominant Alzheimer disease (AD) with onset age below 65 years (presenile AD). We performed a systematic mutation analysis of all coding and 5'-non-coding exons of PS -1 and PS -2 in a population-based epidemiological series of 101 unrelated familial and sporadic presenile AD cases. The familial cases included 10 patients of autosomal dominant AD families sampled for linkage analysis studies. In all patients mutations in the amyloid precursor protein gene ( APP ) had previously been excluded. Four different PS -1 missense mutations were identified in six familial cases, two of which where autosomal dominant cases. Three mutations resulted in onset ages above 55 years, with one segregating in an autosomal dominant family with mean onset age 64 years (range 50-78 years). One PS -2 mutation was identified in a sporadic case with onset age 62 years. Our mutation data provided estimates for PS -1 and PS -2 mutation frequencies in presenile AD of 6 and 1% respectively. When family history was accounted for mutation frequencies for PS -1 were 9% in familial cases and 18% in autosomal dominant cases. Further, polymorphisms were detected in the promoter and the 5'-non-coding region of PS -1 and in intronic and exonic sequences of PS -2 that will be useful in genetic association studies.     

Extraction of cadmium (II) from phosphoric acid media using the di(2-ethylhexyl)dithiophosphoric acid (D2EHDTPA): Feasibility of a continuous extraction-stripping process

The extraction of cadmium from phosphoric media has been studied. The D₂EHDTPA was used as extractant and dodecane as diluent. No third phase was observed in the investigated conditions.A continuous micro-pilot scale mixer-settler was successfully tested for both extraction and stripping. More than 99% extraction rate was obtained in steady-state conditions with a flow rate ratio Aqueous/Organic equal to 1.1. Continuous stripping was performed using HCl 4 M. More than 96% of the cadmium was stripped in one continuous mixer-settler stage for flow rate ratio equal to 0.7. Results were in good agreement with the predicted values based on the McCabe–Thiele method. Experimental mixer-settler stages behave as ideal ones (Murphree efficiency > 98%).An optimal flow sheet is proposed to purify the Wet Phosphoric Acid (WPA) and to recover a relatively concentrated cadmium solution (1 g L^? 1). Two ideal stages operating at phase ratio A/S equal to 5/1 are required for the extraction step leading to a very depleted raffinate (< 0.2 µg L^? 1). For the stripping step, six stages are required (S/A = 5/1). The recovered organic phase contains less than 2 µg L^? 1 and could be recycled in the extraction step.     

Effects of chronic systemic administration of 5-HT on food intake and body weight in rats

The effects of chronic peripheral administration of 5-HT on food intake and body weight was investigated. In normal male Wistar rats, normal female Wistars, obese Zucker males, ovariectomised Wistar females, or normal Wistar males free fed a cafeteria diet, suppression of the creeping weight gain typical of control animals is observed. In females, this effect is not dependent on the local hormonal environment, because intact and ovariectomised females showed similar responses to treatment. One sex difference is that the weight suppressive effect in males is accompanied by an anorectic effect, whereas this anorectic effect is absent in females. Thus, although reduced food intake may partially explain the suppression of weight gain in males, in females it must be due to other, perhaps metabolic, effects. It is possible that these metabolic effects may also occur in males, suggesting one possible explanation of why the effect was typically larger in males than females.     

Effects of chronic stimulation or antagonism of opiate receptors on GH secretion in male and female rats

The present study was undertaken to assess the role of endogenous opioid systems in the sexually dimorphic pattern of growth hormone (GH) secretion. To this end, male rats were treated chronically (6 to 12 h) with morphine and estrogen-exposed, ovariectomized female rats with morphine or naloxone. Chronic morphine exposure of male rats caused a 12-fold increase in basal GH levels and a modest rise in GH pulse frequency. These two changes resulted in a 3-fold increase in both mean GH concentration and total GH secretion over 6 h. In female rats, chronic morphine reduced GH pulse amplitudes but did not significantly affect other parameters of GH secretion. By contrast, chronic naloxone treatment of female rats reduced basal GH levels by 64% without affecting GH pulse amplitudes or pulse frequency. These data suggest that endogenous opioid systems are involved in the regulation of the basal GH secretion in both male and female rats.     

Synergistic extraction of rare earth by mixtures of 2-ethylhexyl phosphoric acid mono-2-ethylhexyl ester and di-（2-ethylhexyl） phosphoric acid from sulfuric acid medium

The extraction of Nd^3＋ and Sm^3＋, including the extraction and stripping capability as well as the separation effect of Nd^3＋ or Sm^3＋, from a sulfuric acid medium, by mixtures of di-（2-ethylhexyl） phosphoric acid （HDEHP, H2A2（0）） and 2-ethylhexyl phosphoric acid mono-2-ethylhexyl ester （HEH/EHP, H2L2（0）） were studied. The distribution ratios and synergistic coefficients of Nd^3＋ and Sm^3＋ in different acidities were also determined. A synergistic extractive effect was found when HDEHP and HEH/EHP were used as mixed extractants for Sm^3＋ or Nd^3＋. The chemical compositions of the extracted complex were determined as Nd.（HA2）2-HL2 and Sm.（HA2）2-HL2. The extraction equilibrium constants, enthalpy change, and entropy change of the extraction reaction were also determined.     

Cortisol secretion rate (CSR) in hypertension and endocrine patients

With the method of isotope dilution the circadian cortisol secretion was determined in 22 patients with hypertonic disease--28.35 mg/24 h, elevated versus CSR in healthy subjects-16.63 mg/24 h. CSR is elevated also in 5 patients with bilateral adenocortical hyperplasia and with suprarenal cancer. The patients with diencephalic syndrome, progressing according to the type of Cushing disease have elevated absolute values of CSR, but recalculated per square meter of body surface and per gram creatineine excreted in 24 h-CSR proved to be normal-about 12 mg/24 h.