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

A target-organ study of the effects of the phthalate ester di-(2-ethylhexyl) phthalate (DEHP) has been conducted in mature male albino ferrets. DEHP treatment caused a loss of body weight when administered as a 1% (w/w) diet for 14 months. Additionally, marked liver enlargement with associated morphological and biochemical changes was observed. These changes consisted of liver cell enlargement, lysosomal changes, dilatation of the endoplasmic reticulum and the depression of a number of marker enzyme activities. The only other tissue observed to be affected by DEHP treatment was the testes where histological evidence of tissue damage was observed in some animals.Studies on the metabolism of [¹⁴C]DEHP in the ferret indicated that the diester was metabolised to derivatives of mono-(2-ethylhexyl) phthalate which were excreted in the urine both unconjugated and as glucuronides.The results obtained have been compared with previous studies in the rat and it is concluded that DEHP is hepatotoxic in both species.     

Effect of di-2-ethylhexyl phthalate (DEHP) on glycogen metabolism in rat liver

Effect of di-2-ethylhexyl phthalate (DEHP) on glycogen contents and certain enzymes of carbohydrate metabolism of rat liver was investigated. A significant decrease in glycogen content of unfasted and an increase in fasted animals was observed. Blood glucose tolerance was reduced and the rate of both glycogenesis and glycogenolysis, as judged by measuring glycogen contents after feeding labelled and unlabelled glucose, was also decreased. Activities of glucose-6-phosphate dehydrogenase, phosphorylase and glucose-6-phosphatase were significantly decreased while activities of fructose-1-6-diphosphate and aldolase remained unaltered. The present results suggest that DEHP affects both glycogenesis and glycogenolysis in rat liver.     

Teratogenicity of di-(2-ethylhexyl)phthalate in mice

Fetotoxicity of di-(2-ethylhexyl)phthalate (DEHP) was studied in a random strain (ddY-Slc♀ × CBA ♂) of mice. A single oral administration of DEHP 0.05 ml/kg on day 7 of gestation resulted in a decrease in body weight of live fetuses, but there were no dead, gross, or skeletal abnormal fetuses. At 0.1 ml/kg and above DEHP decreased fetal body weight and the fetuses were dead or deformed. The fetotoxicity was dose dependent and a straight line Y = 51.9 log X + 61.6 was obtained where Y = the rate of death(%) and X = the dose of DEHP administered (ml/kg). The LD₅₀ and the non-effective aximum dose which induced fetal death was 0.60 ml/kg and 0.065 ml/kg, respectively. The non-effective maximum doses which resulted in gross and skeletal abnormalities were 0.80 and 0.68 ml/kg, respectively.     

The absorption, metabolism and tissue distribution of di(2-ethylhexyl)phthalate in rats

Rats given a single oral dose of [¹⁴C] di(2-ethylhexyl) phthalate [¹⁴C] (DEHP) excreted 42% and 57% of the dose in the urine and faeces respectively in 7 days. A significant proportion (14%) of the dose is excreted in bile. Rats fed 1000 ppm DEHP in the diet for 7 days prior to dosing with [¹⁴C] DEHP excreted 57% and 38% in the urine and faeces respectively in 4 days.When fed continuously to rats at dietary concentrations of 1000 and 5000 ppm, the amount of the ester in liver and abdominal fat rapidly attains a steady-state concentration and there is no evidence of accumulation. When returned to a normal diet, the radioactivity in the liver declined with a half life of 1–2 days while that in fat declined rather more slowly to give a half life of 3–5 days. The relative liver weight increased to a level 50% above normal in rats receiving 5000 ppm DEHP and returned to normal within 1 week after being returned to normal diet.When administered intravenously DEHP is preferentially localised in lung, liver and spleen from where it is eliminated with a half-life of 1–2 days.The hexobarbital sleeping time was reduced by 30–40% in rats following repeated oral administration of DEHP; when the ester was administered intravenously sleeping time was increased by approx. 40%.DEHP is extensively metabolised after oral administration, the principal metabolites being identified as the acid, alcohol and ketone resulting from ω- and (ω-1)-oxidation of mono(2-ethylhexyl) phthalate (MEHP). DEHP is rapidly hydrolysed to the half-ester by pancreatic lipase.     

Interaction of di-(2-ethylhexyl) phthalate with the pharmacological response and metabolic aspects of ethanol in mice

The interactions of di-(2-ethylhexyl) phthalate (DEHP) with the pharmacological response and metabolic aspects of ethanol in mice were investigated at oral doses of DEHP of 1.5, 3.0 and 7.5 g/kg or intraperitoneal doses of 3.7, 7.5 and 18.9 g/kg, administered once or daily for 7 days. A single oral or intraperitoneal administration of DEHP resulted in a significant increase in the ethanol-induced sleeping time, associated with an inhibition of alcohol dehydrogenase activity in liver; the effect of intraperitoneal administration was significant only at the highest dose. The activities of high and low Km aldehyde dehydrogenases in mouse liver were not affected by a single dose of DEHP by either route. Repeated oral doses of DEHP produced significant reductions in the ethanol-induced sleeping time and increases in the activities of alcohol and aldehyde dehydrogenases, whereas repeated intraperitoneal doses of DEHP significantly increased the sleeping time and decreased the activity of alcohol dehydrogenase, without any perceptible effect on the activities of aldehyde dehydrogenases. In vitro studies with mouse liver preparations revealed significant inhibition of alcohol dehydrogenase activity by mono-(2-ethylhexyl) phthalate and 2-ethylhexanol and of high and low Km aldehyde dehydrogenase activities by DEHP and mono-(2-ethylhexyl) phthalate at concentrations ranging from 0.03 to 1.00 mM. In all cases, in vitro enzyme inhibition by mono-(2-ethylhexyl) phthalate was most pronounced.     

The absence of testicular atrophy and in vivo and in vitro effects on hepatocyte morphology and peroxisomal enzyme activities in male rats following the administration of several alkanols

Previous studies have shown that ethylhexanol (2-EH) and its oxidation products, but not n-hexanol, produce hepatomegaly, peroxisomal proliferation and hypotriglyceridaemia. In the present studies we have confirmed that at 1 mmol/kg doses, neither the linear nor branched chain alcohols induce testicular atrophy, hepatomegaly, peroxisome proliferation or hypolipidaemia. In vivo, neither the free alcohols nor their metabolic products seem to be responsible for the activity of the parent plasticiser. The released monoesters are probably the more potent metabolic products responsible for the hepatomegaly, peroxisomal proliferation and hypolipidaemia. This contention is supported by the in vitro hepatocyte data which demonstrate the induction of peroxisomal oxidative enzymes by MEHP whereas the alcohols were without effects.     

Serum triglyceride and cholesterol contents in male rats receiving diets containing plasticizers and analogues of the ester 2-ethylhexanol

Male rats were administered diets containing the plasticizers di-(2-ethylhexyl) adipate and di-(ethyl)phthalate, 2-ethylhexanol, hexanol, 2-ethylhexanoic acid, hexanoic acid, 2-ethylhexyl aldehyde, and hexyl aldehyde for 3 weeks, and serum triglyceride and cholesterol values were determined. Only those compounds which had been found to produce a proliferation of hepatic peroxisomes produced a decrease in both serum lipids.     

Effect of di-2-ethylhexyl phthalate on the activity of succinic dehydrogenase and adenosine triphosphatase of some vital organs of rat.

Succinic dehydrogenase (SDH) and adenosine triphosphatase (ATPase) activity measured 21 days after 3 intraperitoneal injections of di-2-ethylhexyl phthalate (DEHP) was decreased in heart, lung and kidney while it remained unaltered in brain. Under in vitro conditions, only SDH was sensitive to DEHP, degree of inhibition being concentration-dependent. DEHP solubilized with Tween 80 inhibited the SDH activity significantly at small concentrations of 5 to 25 ppm. ATPase activity was not modified.     

Disposition of orally administered di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate in the rat

The dispositon of di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) was studied in the rat. Three hours after a single oral dose of DEHP (2.8 g/kg), plasma concentrations of 8.8±1.7 g/ml DEHP and 63.2±8.7 g/ml MEHP were reached. MEHP levels declined with a half-life of 5.2±0.5 h. The ratio of the area under the plasma concentration-time curve of MEHP to that of DEHP was 16.1±6.1. When ¹⁴CDEHP was administered, 19.3±3.3% of the radioactivity was excreted in the urine within 72 h, the rest being excreted in the faeces. The urinary excretion rate of total radioactivity declined with a half-life of 7.9±0.5 h. Single administration of MEHP (0.4 g/kg) resulted in plasma concentrations of 84.1±14.9 g/ml 3 h after dosing; the half-life of MEHP was 5.5±1.1 h. Multiple dosing with DEHP (2.8 g/kg/day) for 7 consecutive days produced no accumulation of DEHP or MEHP in plasma.     

Di-(2-ethylhexyl) phthalate enhances the release of lysosomal enzymes from alveolar macrophages during phagocytosis

Lung tissue damage, histologically similar to protease induced lung lesions, has been previously demonstrated in animals exposed to the plasticizer, di-(2-ethylhexyl)phthalate (DEHO). In an attempt to identify the mechanism responsible for this damage, we have examined the effect of DEHP on alveolar macrophages. Serum solubilized DEHP has a significant effect on both the phogocytosis of latex particles and lysosomal enzyme released from rabbit alveolar macrophages. Pre-exposure to 2 mg% DEHP caused a 2-fold increase in the rate of phagocytosis and an 8-fold and 10-fold increase, respectively, in the release of the lysosomal hydrolases β-glucuronidase and acid phosphatase. Although exposure to 2 mg% DEHP caused an 8-fold increase in in vitro cell death, pre-exposure to DEHP had only minimal effect on death during subsequent cell culture, as indicated by measurement of dye exclusion and the release of the cytosolic enzyme lactate dehydrogenase. The relationship between the DEHP induced increase in lysosomal enzyme release from alveolar macrophages and the pathological and histological effects of DEHP on pulmonary tissue is discussed, particularly with respect to patients receiving multiple blood transfusions.     

邻苯二甲酸二乙基己基酯对胎鼠肺发育的抑制作用

目的探讨孕大鼠染毒邻苯二甲酸二乙基己基酯(DEHP)对胎鼠肺发育的抑制作用及其可能机制。方法 Sprague-Dawley大鼠受孕后第12天每天ig给予DEHP0,10,100和750mg.kg-1,至自然分娩。第1天每窝随机取自然分娩仔鼠3只,测定体质量;光镜观察肺组织病理学改变及测定辐射状肺泡计数(RAC)和肺间质比例,免疫组化法检测基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶组织抑制剂-2(TIMP-2)和血管内皮生长因子(VEGF)的表达。结果与正常对照组相比,DEHP组仔鼠体质量明显下降(P<0.01)。光镜下仅DEHP750mg.kg-1组可见肺间质增厚,间质细胞增多,肺泡数目减少,RAC减小,肺间质比例增大(P<0.05)。与正常对照组比较,DEHP组VEGF表达差异无统计学意义;DEHP10,100和750mg.kg-1组MMP-2表达和MMP-2/TIMP-2值明显高于正常对照组〔MMP-2分别为0.099±0.009,0.124±0.008,0.140±0.010vs0.091±0.011(P<0.01);MMP-2/TIMP-2分别为1.079±0.074,1.447±0.077,1.704±0.084vs0.994±0.079(P<0.01)〕。结论孕鼠染毒DEHP后对胚胎生长和肺发育有抑制作用。DEHP抑制胎鼠肺发育的机制可能与MMP-2的过度表达以及MMP-2/TIMP-2平衡失调有关。     

Skin permeation and metabolism of di(2-ethylhexyl) phthalate (DEHP)

Phthalates are suspected to be endocrine disruptors. Di(2-ethylhexyl) phthalate (DEHP) is assumed to have low dermal absorption; however, previous in vitro skin permeation studies have shown large permeation differences. Our aims were to determine DEHP permeation parameters and assess extent of skin DEHP metabolism among workers highly exposed to these lipophilic, low volatile substances.     

Di(2-ethylhexyl)phthalate (DEHP) metabolites in human urine and serum after a single oral dose of deuterium-labelled DEHP

Human metabolism of di(2-ethylhexyl)phthalate (DEHP) was studied after a single oral dose of 48.1 mg to a male volunteer. To avoid interference by background exposure the D4-ring-labelled DEHP analogue was dosed. Excretion of three metabolites, mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono(2-ethylhexyl)phthalate (MEHP), was monitored for 44 h in urine and for 8 h in serum. Peak concentrations of all metabolites were found in serum after 2 h and in urine after 2 h (MEHP) and after 4 h (5OH-MEHP and 5oxo-MEHP). While the major metabolite in serum was MEHP, the major metabolite in urine was 5OH-MEHP, followed by 5oxo-MEHP and MEHP. Excretion in urine followed a multi-phase elimination model. After an absorption and distribution phase of 4 to 8 h, half-life times of excretion in the first elimination phase were approximately 2 h with slightly higher half-life times for 5OH- and 5oxo-MEHP. Half-life times in the second phase—beginning 14 to 18 h post dose—were 5 h for MEHP and 10 h for 5OH-MEHP and 5oxo-MEHP. In the time window 36 to 44 h, no decrease in excreted concentrations of 5OH- and 5oxo-MEHP was observed. In the first elimination phase (8 to 14 h post dose), mean excretion ratios of MEHP to 5oxo-MEHP and MEHP to 5OH-MEHP were 1 to 1.8 and 1 to 3.1. In the second elimination phase up to 24 h post dose mean excretion ratios of MEHP to 5oxo-MEHP to 5OH-MEHP were 1 to 5.0 to 9.3. The excretion ratio of 5OH-MEHP to 5oxo-MEHP remained constant through time at 1.7 in the mean. After 44 h, 47% of the DEHP dose was excreted in urine, comprising MEHP (7.3%), 5OH-MEHP (24.7%) and 5oxo-MEHP (14.9%).     

Effects of di-(2-ethylhexyl) phthalate (DEHP) on behavior and dopamine signaling in zebrafish (Danio rerio)

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer, also known as a developmental toxicant, but its neurobehavioral toxicity remains elusive. This study evaluated the neurobehavioral toxicity and its possible mechanism in larval zebrafish. Embryos at gastrula period (~6 h post fertilization, hpf) were exposure to DEHP (0, 1, 2.5, 5 and 10 mg/L) for 7 days. Spontaneous tail movement in embryos and swimming activity in larvae were monitored. Alterations in the mRNA expression of genes involved in dopamine signaling and apoptosis pathway were assessed. In situ apoptotic cells were assessed by Acridine orange staining, and oxidative damage were measured using enzymatic assay. The behavior results showed that DEHP inhibited spontaneous tail movement and decreased locomotor activities in the light/dark behavioral test. Meanwhile, behavioral changes were accompanied by increased apoptosis and malondialdehyde (MDA) content, decreased superoxide dismutase (SOD) activity and dopamine (DA) content, and perturbed the expression of genes associated with the synthesis (th), reuptake (dat) and metabolism (mao) of DA, with dopamine receptors (DRs), and with the apoptosis pathway (p53, bax, bcl2, caspase-3, caspase-8, caspase-9). The findings will help to illuminate the possible neurobehavioral toxicity mechanisms of organism exposure to DEHP.     

Assessment of the teratogenicity of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in mice

Di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) were administered PO or IP to pregnant ICR mice at varying doses on days 7, 8, and 9 of gestation. In groups given DEHP orally, resorptions and malformed fetuses increased significantly at 1,000 mg/kg. Fetal weights were also significantly suppressed. Anterior neural tube defects (anencephaly and exencephaly) were the malformations most commonly produced. No teratogenic effects were revealed by IP doses of DEHP and PO or IP doses of MEHP, although high doses were abortifacient and lethal to pregnant females. Thus DEHP is highly embryotoxic and teratogenic in mice when given PO but not IP. The difference in metabolism, disposition, or excretion by the route of administration may be responsible for the difference in DEHP teratogenicity. Although MEHP is a principal metabolite of DEHP and is several times more toxic than DEHP to adult mice, it seems that MEHP and its metabolites are not teratogenic in ICR mice.     

Chronic toxicity of di(2-ethylhexyl)phthalate in rats.

Fischer 344 rats were treated with 0, 100, 500, 2500, or 12,500 ppm di(2-ethylhexyl)phthalate (DEHP) in the diet for up to 104 weeks. Blood and urine were analyzed at weeks 26, 52, 78, and 104 from 10 animals per sex per group. Survival was slightly but not statistically reduced for rats receiving 12,500 ppm DEHP. Body weights and food consumption were significantly reduced for rats receiving the highest dose level of DEHP and occasionally for the male 2500-ppm group. BUN and albumin were significantly higher and globulin lower at nearly every sampling interval for the 12,500-ppm group compared with the controls. There was an increase in the mean activities of AST and ALT at 104 weeks, but no statistically significant differences were seen. Erythrocyte count, hemoglobin, and hematocrit values for the 12,500-ppm group were significantly lower than controls at nearly every sampling interval. No other differences in hematology were seen. No toxicologically significant changes were observed in urinalysis. At termination, relative lung weights for the 2500- and 12,500-ppm male groups of rats were significantly higher than for the controls. Absolute and relative liver and kidney weights for the 2500- and 12,500-ppm male rats, and liver weights for 12,500-ppm female rats were higher compared with the controls. Absolute and relative testes weights for the 12, 500-ppm male rats were lower compared with the controls. All organs were examined for histopathology. The incidence of hepatocellular lesions has been reported separately and correlated with the induction of peroxisomal enzyme activity (David et al., 1999). A dose level of 500 ppm was the NOEL for peroxisome proliferation. Bilateral aspermatogenesis in the testes, castration cells in the pituitary gland, spongiosis hepatis, and pancreatic acinar cell adenoma were observed for 12,500-ppm male rats. Aspermatogenesis and spongiosis hepatis were observed for 2500-ppm male rats, and aspermatogenesis was seen at 500 ppm. DEHP exposure exacerbated age-, species- or strain-related lesions such as mineralization of the renal papilla and chronic progressive nephropathy in male rats. Kupffer cell pigmentation and renal tubule pigmentation were seen in male and female 12,500-ppm rats. The increased incidence of spongiosis hepatis correlated with increased palmitoyl CoA oxidase activity, but the incidence of pancreatic acinar cell adenoma was increased only at the highest dose level of 12,500 ppm. These lesions, although typical of those seen with other peroxisome proliferators, may respond differently depending on the potency of the peroxisome proliferator. A dose level of 500 ppm (28.9-36.1 mg/kg/day) was considered to be the NOAEL.     

A dose-response study following in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP): effects on androgenic status, developmental landmarks and testicular histology in male offspring rats

An extensive dose-response study following in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP) was conducted. A wide range of low and high DEHP doses were tested. Reproductive effects were evaluated on male offspring rats. Female Wistar rats were treated daily with DEHP and peanut oil by gavage from gestation day 6 to lactation day 21 at doses of 0.015, 0.045, 0.135, 0.405 and 1.215 mg DEHP/kg body weight (bw)/day (low doses) and at 5, 15, 45, 135 and 405 mg DEHP/kg bw/day (high doses). Nipple retention and reduced anogenital distance, both sensitive markers of anti-androgenic effects during development, were only seen in males exposed to the highest dose (405 mg/kg/day). Delayed preputial separation was observed in animals exposed to 15 mg DEHP/kg/day and higher doses. Histopathological examination of the testis on postnatal days (PNDs) 1 and 22 revealed changes at 135 and 405 mg DEHP/kg/day. The most prominent finding on PND 1 was the presence of bi- and multinucleated gonocytes. On PND 22 signs of reduced germ cell differentiation in seminiferous tubules of exposed animals were observed. Testis weight on PND 22 was significantly increased at 5, 15, 45 and 135 mg/kg/day, an effect that qualitatively differs from exposure to higher doses. The current results show that DEHP acts as an anti-androgen at a high dose exposure (405 mg/kg/day). However, these results also indicate that other subtle developmental effects occur at lower DEHP doses.     

Blood burden of di(2-ethylhexyl) phthalate and its primary metabolite mono(2-ethylhexyl) phthalate in pregnant and nonpregnant rats and marmosets

A comparison of the dose-dependent blood burden of di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate (MEHP) in pregnant and nonpregnant rats and marmosets is presented. Sprague-Dawley rats and marmosets were treated orally with 30 or 500 mg DEHP/kg per day, nonpregnant animals on 7 (rats) and 29 (marmosets) consecutive days, pregnant animals on gestation days 14-19 (rats) and 96-124 (marmosets). In addition, rats received a single dose of 1000 mg DEHP/kg. Blood was collected up to 48 h after dosing. Concentrations of DEHP and MEHP in blood were determined by GC/MS. In rats, normalized areas under the concentration-time curves (AUCs) of DEHP were two orders of magnitude smaller than the normalized AUCs of the first metabolite MEHP. Metabolism of MEHP was saturable. Repeated DEHP treatment and pregnancy had only little influence on the normalized AUC of MEHP. In marmosets, most of MEHP concentration-time courses oscillated. Normalized AUCs of DEHP were at least one order of magnitude smaller than those of MEHP. In pregnant marmosets, normalized AUCs of MEHP were similar to those in nonpregnant animals with the exception that at 500 mg DEHP/kg per day, the normalized AUCs determined on gestation days 103, 117, and 124 were distinctly smaller. The maximum concentrations of MEHP in blood of marmosets were up to 7.5 times and the normalized AUCs up to 16 times lower than in rats receiving the same daily oral DEHP dose per kilogram of body weight. From this toxicokinetic comparison, DEHP can be expected to be several times less effective in the offspring of marmosets than in that of rats if the blood burden by MEHP in dams can be regarded as a dose surrogate for the MEHP burden in their fetuses.     

Effects of di-(2-ethylhexyl) phthalate on the hypothalamus-pituitary-ovarian axis in adult female rats

Di-(2-ethylhexyl) phthalate (DEHP), an environmental endocrine disruptor, is widely present in the environment and some products with phthalate plasticizer. It has become a serious problem in recent years. The effect of DEHP on female reproductive system is still not well-studied. This study was to investigate the effects of DEHP on hypothalamus-pituitary-ovarian axis in adult female rats. Compared with control rats, the DEHP-treated rats showed: (1) lower body weight; (2) lower organ coefficient of ovary; (3) higher GnRH level in the hypothalamus; (4) higher mRNA and protein levels of GnRHR in the pituitary; and (5) lower serum sex hormone levels. Our data reveal that DEHP exposure may lead to the disruption of estrogen biosynthesis pathways in female rats and imbalance of hypothalamus-pituitary-ovarian axis. DEHP may impose negative influence on the development and function of the reproductive system in female rats.