Effects of endocrine disrupting chemicals on in vitro global DNA methylation and adipocyte differentiation

Recent studies suggest that endocrine disrupting chemicals (EDCs) may form a risk factor for obesity by altering energy metabolism through epigenetic gene regulation. The goal of this study is to investigate the effects of a range of EDCs with putative obesogenic properties on global DNA methylation and adipocyte differentiation in vitro. Murine N2A and human SK-N-AS neuroblastoma cells and murine preadipocyte fibroblasts (3T3-L1) were exposed to tributyltin (TBT), diethylstilbestrol (DES), bisphenol A (BPA), 2,3,7,8-tetrachlorodibenzo-[p]-dioxin (TCDD), 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB-153), hexachlorobenzene (HCB), hexabromocyclododecane (HBCD), 2,2′,4,4′-tetrabrominated diphenyl ether (BDE-47) , perfluorinated octyl acid (PFOA) and perfluorinated octyl sulfonate (PFOS). A modest decrease in global DNA methylation was observed in N2A cells exposed to 10 μM DES, BPA, TCDD, BDE-47, PCB-153 and 1 μM HCB, but no changes were found in the human SK-N-AS cells. We reveal for the first time that BDE-47 increases adipocyte differentiation in a dose-dependent manner (2.5–25 μM). Adipocyte differentiation was also enhanced by TBT (?10 nM) and BPA (>10 μM) and inhibited by TCDD (?0.1 nM). The other chemicals showed either modest or no effects on adipocyte differentiation at the concentrations tested (PFOA, PFOS and HBCD at 10 μM; PCB-153, 3.4 μM and HCB, 1 μM). This study demonstrates that selected EDCs can induce functional changes in murine adipocyte differentiation in vitro which are accompanied by decreased global DNA methylation.     

Roles of endoplasmic reticulum stress,apoptosis and autophagy in 2,2′,4,4′-tetrabromodiphenyl ether-induced rat ovarian injury

Endocrine disruptor 2,2′,4,4′-tetrabromodiphenylether (PBDE-47) can harm the female reproductive system. Recent studies showed that PBDE-47 neurotoxicity is associated with endoplasmic reticulum stress (ERS); however, the role of ERS in PBDE-47-induced ovarian injury is unclear. New-born female Sprague-Dawley rats were orally exposed to PBDE-47 (1, 5, or 10 mg/kg bw) on postnatal day 10. An additional 10 mg/kg bw PBDE-47 group was given the ERS inhibitor 4-PBA intraperitoneally for three weeks beginning on postnatal day 8. At 2 months of age, PBDE-47 exposure significantly reduced the ovarian coefficients, increased the expression of ERS and autophagy markers, including GRP78, IRE1, Caspase-12, Beclin1, LC3 and P62. In the 10 mg/kg bw PBDE-47 group, PARP and Caspase-3 were markedly activated, indicative of apoptosis. These were accompanied by histopathological damage. Intriguingly, 4-PBA attenuated all these effects. Thus, these results suggest that ERS plays a vital role in PBDE-47-induced ovarian injury by regulating autophagy and apoptosis.     

Dietary exposure to polybrominated diphenyl ether 47 (BDE-47) inhibits development and alters thyroid hormone-related gene expression in the brain of Xenopus laevis tadpoles

Few studies have investigated the thyroid-disrupting effects of polybrominated diphenyl ethers (PBDEs) across multiple levels of biological organization in anurans, despite their suitability for the screening of thyroid disruptors. Therefore, the present study evaluated the effects of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) on development, thyroid histology and thyroid hormone-related gene expression in Xenopus laevis exposed to 0 (control), 50 (low), 500 (medium) or 5000 μg BDE-47/g food (high) for 21 days. Only the high dose of BDE-47 hindered growth and development; however, thyroid hormone-associated gene expression was downregulated in the brains of tadpoles regardless of dose. These results show that BDE-47 disrupts thyroid hormone signaling at the molecular and whole-organism levels and suggest that gene expression in the brain is a more sensitive endpoint than metamorphosis. Furthermore, the altered gene expression patterns among BDE-47-exposed tadpoles provide insight into the mechanisms of PBDE-induced thyroid disruption and highlight the potential for PBDEs to act as neurodevelopmental toxicants.     

Cytotoxicity and apoptosis induction on RTG-2 cells of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and decabrominated diphenyl ether (BDE-209)

Recently, the environmental residues of polybrominated diphenyl ethers (PBDEs) have markedly increased. In particular, the levels of certain PBDE congeners in fish have raised concern regarding potential risks associated with dietary PBDEs exposures. However, little is known regarding PBDE-mediated cell injury in relevant in vitro fish cell models. In this study, the cytotoxic effects of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and decabrominated diphenyl ether (BDE-209) on RTG-2 cells were investigated. RTG-2 cells were incubated with different concentrations of BDE-47 and BDE-209 (1–100 μM) for 72 h, and a set of bioassays were conducted to measure: cell viability (evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and neutral red (NR) uptake), lactate dehydrogenase (LDH) leakage, reactive oxygen species (ROS) formation and cell apoptosis. The results showed that BDE-47 and BDE-209 inhibited the cells viability, increased LDH leakage, and induced cell apoptosis in time and concentration-dependent manner. All significant effects were observed at concentrations of 12.5 μM and above for BDE-47 and 25 μM and above for BDE-209 (P < 0.05). At the concentration of 100 μM BDE-47 and BDE-209, the cell viability of the exposed cells dropped to about 40% and 50% of the control, and the apoptotic rates were 52.6% and 34.6%, respectively. After 12 h exposure, a concentration-dependent increases of BDE-47 and BDE-209 (12.5–100 μM) in ROS formation were observed. Collectively, the results of cell viability, LDH leakage, cell apoptosis and ROS formation demonstrated that the toxic mechanism of PBDEs on RTG-2 might be mediated by oxidative stress.     

Effects of perchlorate on BDE-47-induced alteration thyroid hormone and gene expression of in the hypothalamus–pituitary–thyroid axis in zebrafish larvae

To investigate the effects of perchlorate on thyroid hormone disturbances induced by 2,2′,4′,4-tetrabromodiphenyl ether (BDE-47) via thyroid hormone (TH)-mediated pathways, zebrafish embryos were exposed to a combination of BDE-47 and PER from the time of fertilisation to 14 d (dpf). The whole-body content of TH and the expression of genes and proteins related to the hypothalamic–pituitary–thyroid (HPT) axis were analysed. Co-exposure to BDE-47 and PER decreased the body weight and increased malformation rates relative to the effects of exposure to only BDE-47. Compared with the exposure to BDE-47 alone, the exposure to a combination of BDE-47 (10 μg/L) and PER (3.5 mg/L) significantly up-regulated the expression of genes involved in TH synthesis (NIS and Nkx2.1a) and significantly down-regulated the expression of genes related to the regulation of the HPT axis (CRH and TSHβ). The expression of TG at the gene and protein levels was significantly up-regulated, but the expression of TTR was significantly down-regulated in the co-exposures relative to BDE-47 treated alone. In addition, the larger reduction in the T4 level resulting from exposure to the mixture of BDE-47 and PER demonstrated that PER enhanced the thyroid-disruptive effects of BDE-47. These results help to elucidate the complicated chemical interactions and the molecular mechanism of action of these two TH disruptors.     

An antioxidant,N,N′-diphenyl-p-phenylenediamine (DPPD), affects labor and delivery in rats: A 28-day repeated dose test and reproduction/developmental toxicity test

A 28-day repeated dose toxicity test and reproduction/developmental toxicity test for N,N′-diphenyl-p-phenylenediamine (DPPD) were conducted in [Crl:CD(SD)] SPF rats. Male and female rats were dosed with DPPD by gavage for 28 days at 0, 100, 300, or 1000 mg/kg bw/day or for a total of 42–46 days at 0, 8, 50, or 300 mg/kg bw/day. No significant adverse effects were observed in the repeated dose toxicity study up to 1000 mg/kg bw/day in both sexes. In the reproduction/developmental toxicity study, two females showed piloerection, hypothermia, and pale skin; one died and the other showed dystocia on day 23 of pregnancy at 300 mg/kg bw/day. Another female delivered only three live pups at 300 mg/kg bw/day. A significantly prolonged gestation period was observed at 50 and 300 mg/kg bw/day. The NOAELs of repeated dose toxicity and reproduction/developmental toxicity were considered to be 1000 and 8 mg/kg bw/day, respectively.     

Long-term safety of α-lipoic acid (ALA) consumption: A 2-year study

α-Lipoic acid (ALA) (CAS RN 1077-28-7), also referred to as thioctic acid, has been demonstrated to exhibit strong anti-oxidant properties. In order to test the long-term toxicity of ALA, groups of 40–50 male and female, 5–6-week-old, Sprague–Dawley rats were subjected to oral administration of 20, 60, or 180 mg/kg body weight (bw)/day ALA for 24 months. There was no significant difference between control animals and treated animals at 20 or 60 mg/kg bw/day with respect to body weight gain, food consumption, behavioural effects, haematological and clinical chemistry parameters, and gross and histopathological findings. In all treatment groups, mortality was slightly lower as compared to the control. The absolute weights of the heart (high-dose males), thymus (high-dose males), and left adrenal (mid-dose males), liver (high-dose females), and lungs (high-dose females) were decreased in comparison to controls. These changes were of no toxicological significance. The only notable finding in rats of both sexes dosed at 180 mg/kg bw/day was a reduction in food intake relative to the controls and a concomitant decrease in body weight. This decrease in body weight led to significant differences between the control and high-dose rats with respect to the absolute weights of certain organs. However, no gross or histopathological changes were associated with these findings. The no-observed-adverse-effect level (NOAEL) is considered to be 60 mg/kg bw/day.     

Effect of developmental dioxin exposure on methylation and expression of specific imprinted genes in mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disruptor affecting the reproductive system in humans. The aim of this study was to evaluate the effects of TCDD administered to pregnant mice at two different doses (2–10 ng/kg/day), on imprinted genes in the male offspring. The degree of methylation and the mRNA expression of Snrpn, Peg3 and Igf2r were analyzed in the sperm, skeletal muscle and liver. TCDD administration (10 ng/kg/day) decreased the sperm count in the male offspring. It did not affect methylation but increased mRNA expression of Snrpn, Peg3, Igf2r and Air ncRNA. In muscle and liver, TCDD (10 ng/kg/day) induced increases in methylation and decreases in mRNA expression of Igf2r. These results show that the robust effects of TCDD on the mRNA expression of Snrpn, Peg3 and Igf2r genes in the sperm and of Igf2r in the muscle and liver are unrelated to changes in methylation in their respective genes.     

Developmental toxicity of brominated flame retardants,tetrabromobisphenol A and 1,2,5,6,9,10-hexabromocyclododecane,in rat offspring after maternal exposure from mid-gestation through lactation

To evaluate developmental exposure effects of two brominated flame retardants, tetrabromobisphenol A (TBBPA) and 1,2,5,6,9,10-hexabromocyclododecane (HBCD), pregnant Sprague–Dawley rats were administered either chemical at doses of 100, 1000 or 10,000 ppm in a soy-free diet from gestation day 10 until the day 20 after delivery. Offspring exposed to TBBPA showed dose-unrelated slight decreases of serum triiodothyronine (T₃) concentration at postnatal day 20, and there was no evidence of hypothyroidism-related neuronal mismigration and impaired oligodendroglial development as judged by morphometric analyses of NeuN-immunoreactive neuronal distribution in the hippocampal CA1, and area of corpus callosum as well as density of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase)-immunoreactive oligodendrocytes in the cingulate deep cortex at the adult stage. On the other hand, HBCD exerted a weak hypothyroidism evident with increases in thyroid weight, thyroid follicular cell hypertrophy and serum concentrations of thyroid-stimulating hormone as well as decreases of serum T₃ concentrations in offspring at 10,000 ppm at weaning. Increased thyroid weights and decreased serum T₃ concentrations were also observed in the adult stage from 1000 ppm. With regard to the effect on brain development, HBCD reduced density of CNPase-positive oligodendrocytes at 10,000 ppm, suggesting an impaired oligodendroglial development. Results thus suggest that TBBPA did not exert developmental brain effects, while HBCD did, and 100 ppm was determined to be the no-observed-adverse-effect level of HBCD from changes in thyroid parameters at the adult stage by maternal exposure, translating into 8.1–21.3 mg/kg-d.     

90-Day dietary toxicity study with esterified propoxylated glycerol (EPG) in Micropigs

The subchronic (90-day) toxicity of esterified propoxylated glycerol (EPG) was assessed in micropigs. Animals (5/sex/group) received feed containing 5%, 10%, and 17% EPG, mixed accordingly throughout the study to deliver 1.5, 3, and 5 g/kg bw/day of EPG, respectively. Corn oil served as the vehicle control (0 g/kg bw/day). Subsets of animals were evaluated at Week 6; the remainder between Weeks 12 and 14. With the exception of liver and serum vitamin levels, statistically significant difference between control and EPG groups were seen sporadically, and with no apparent connection to treatment and/or no consistency across time intervals. EPG intakes of 3 and 5 g/kg bw/day, but not at 1.5 g/kg bw/day were associated with significantly lower serum 25-OH vitamin D levels. Serum total vitamin D levels were significantly lower across all EPG groups. There were also trends toward lower levels of liver vitamins A and E among EPG-treated animals, but the effects were less consistent. The effects on vitamin levels observed in EPG-treated animals were not accompanied by any signs of vitamin deficiency (e.g., effects on growth, clinical signs, or clinical pathology), and might have been related to the larger mass of EPG acting as a lipid “sink” during transit in the gastrointestinal tract.     

Estrogenic followed by anti-estrogenic effects of PCBs exposure in juvenil fish (Spaurus aurata)

Vitellogenin (Vtg) is a phospho-lipo-glycoprotein produced by oviparous animals in response to estrogen receptor (ER) binding. The presence of Vtg in juvenile and male fish liver and plasma has been used as biomarker to evaluate levels of environmental contaminants as dioxin and PCBs. Interaction of dioxins and PCBs with aryl hydrocarbon receptor (AhR) may affect reproduction by recruitment of estrogen receptor α (ERα). The aim of this study was to investigate the effects of PCB-126, a co-planar PCB prototypical AhR agonist, and of PCB-153, a non-coplanar PCB lacking dioxine-like activity, on Vtg expression in young fish (Spaurus aurata) after a 12 or 24 h exposure to PCBs as well as 48 h following PCBs removal. Vtg expression was evaluated by immunohistochemistry and by Western-blot analysis. Our results showed an increased Vtg expression following PCBs administration, with a maximum level after 12 h of exposure to either PCB-126, PCB-153 or a mixture of both PCBs. Following this estrogenic activity, an anti-estrogenic activity was detected after 24 h of incubation with PCB-126 (alone or mixed with PCB-153), suggested by a decrease in Vtg expression likely through AhR, as a consequence of a hypothetic defence mechanism to endogenous or exogenous ligands.     

Life-cycle exposure to BDE-47 results in thyroid endocrine disruption to adults and offsprings of zebrafish (Danio rerio)

2,2,4′,4′-Tetrabromodi-phenyl ether (BDE-47) is predominantly concentrated in humans and wildlife and disturbs thyroid hormone homeostasis. The purpose of this study was to characterize the thyroid endocrine disruption induced by life-cycle exposure to BDE-47 in adults and offspring of zebrafish (Danio rerio). We exposed zebrafish embryos at the blastula stage to different concentrations of BDE-47 (1, 5, and 10 μg/L). Exposure duration was 180 days until fish reached adulthood. In F0 larvae, exposure decreased survival and increased malformations at 4 dpf. Thyroid hormone concentrations did not differ significantly between the F0 larvae and controls. All exposures significantly up-regulated expression of tshß, pa8, ugt1 and tg and down-regulated ttr. Significant up-regulation of dio2 and crh was observed in the 10 μg/L BDE-47 group. There was no significant difference in the growth and somatic index between F0 adults and controls. BDE-47 (10 μg/L) significantly decreased whole-body content of thyroxine (T4) but significantly increased triiodothyronine (T3) in both sexes. All exposures up-regulated expression of crh, tshß, pa8, ugt1 and tg and down-regulated ttr. Exposure to 10 μg/L BDE-47 significantly up-regulated dio2 and ugt1 in both sexes. BDE-47 exposure (5 and 10 μg/L) significantly increased the activity of pethoxy-resorufin-O-deethylase and UDP-glucuronosyl transferase. BDE-47 (10 μg/L) significantly increased activity of ethoxy- and methoxy-resorufin-O-deethylase. In F1 offspring without continued BDE-47 (10 μg/L) treatment, T4 significantly decreased and T3 increased. T4 was further decreased and T3 was further increased with continued BDE-47 treatment. Continued BDE-47 exposure decreased hatching and increased malformation compared with those without BDE-47 exposure. Expression of crh, tshß, dio2, pa8, ugt1 and tg was significantly up-regulated without BDE-47 exposure and with continued exposure. With continued BDE-47 exposure, dio1 was significantly up-regulated and ttr was significantly down-regulated. All the genes showed clear differences between continued exposure to 10 μg/L BDE-47 and without BDE-47 exposure. These results suggest that parental exposure to BDE-47 results in thyroid endocrine disruption in adults and offspring.     

Prenatal developmental toxicity study of the basic rubber accelerator, 1,3-di-o-tolylguanidine,in rats

Pregnant rats were given 1,3-di-o-tolylguanidine (DTG) by gavage at 0, 10, 20 or 40 mg/kg bw/day on days 6–19 of pregnancy and the pregnancy outcome was determined on day 20 of pregnancy. At 40 mg/kg bw/day, deaths were observed in four out of 24 females. The incidences of females showing mydriasis at 20 and 40 mg/kg bw/day and showing decreased locomotor activity at 40 mg/kg bw/day were significantly increased. Alopecia, bradypnea, prone position and tremor were also observed at 40 mg/kg bw/day. The maternal body weight gain at 20 and 40 mg/kg bw/day and food consumption at 40 mg/kg bw/day were significantly reduced. A significantly decreased weight of the gravid uterus, increased incidence of postimplantation loss, decreased number of live fetuses, and lowered weights of fetuses and placentae were found at 40 mg/kg bw/day. The incidences of the total number of fetuses with external malformations at 40 mg/kg bw/day and with skeletal malformations at 20 and 40 mg/kg bw/day were significantly increased. Significantly higher incidences of fetuses with brachydactyly and short tail and defects of caudal vertebrae, phalanges and metacarpals were observed at 40 mg/kg bw/day. Delayed ossification was also noted at 40 mg/kg bw/day. The data indicate that DTG is teratogenic at maternal toxic doses and the NOAELs of DTG for maternal and developmental toxicity are 10 mg/kg bw/day in rats.     

Phenolic acid protects of renal damage induced by ochratoxin A in a 28-days-oral treatment in rats

The present study aimed to characterize the chlorogenic acid (ChlA) capacity to reverse the toxic effects induced by ochratoxin A (OTA) in a subacute toxicity test in rats. Male Wistar rats were fed orally by gavage for 28 days with OTA (0.4 mg/kg bw/day), ChlA (5 mg/kg bw/day) or the combination OTA (0.4 mg/kg bw/day) + ChlA (5 mg/kg bw/day). No deaths, no decrease in feed intake or body weight in any experimental group were recorded. The negative control group and the animals treated with ChlA alone showed no changes in any parameters evaluated. In OTA-treated group significant changes such as decrease in urine volume, proteinuria, occult blood, increase in serum creatinine values; decrease in absolute and relative kidney weight and characteristics histopathological lesions that indicated kidney damage were observed. However, limited effect on oxidative stress parameters were detected in kidneys of OTA-treated group. Animals treated with the combination OTA + ChlA were showed as negative control group in the evaluation of several parameters of toxicity. In conclusion, ChlA, at given concentration, improved biochemical parameters altered in urine and serum and pathological damages in kidneys induced by OTA exposure, showing a good protective activity, but not by an apparent antioxidant mechanism.     

Effect of natamycin on cytochrome P450 enzymes in rats

Natamycin is a polyene macrolide antibiotic widely used in the food industry as a feed additive to prevent mold contamination of foods. There are many contradictory results on the genotoxic effects of macrolides which could suggest a potential risk for humans. In the present study, the effects of natamycin on the activities of some drug metabolizing enzymes in rat liver microsomes were determined in vivo. Rats were treated orally with natamycin at doses of 0.3, 1, 3 and 10 mg/kg body weight (bw)/day for 6 days. Determinations of cytochrome P450 (CYP) enzyme activities were carried out in hepatic microsomes isolated from rats treated. The activities of CYP2E1, CYP1A1/2 CYP2B1/2 and CYP4A1/2 enzymes significantly decreased after treatment with 1, 3 and 10 mg/kg bw/day, in a dose-dependent manner as compared to control. This effect was not observed after natamycin treatment at dose of 0.3 mg/kg bw/day. Our results suggest that natamycin may not potentiate the toxicity of many xenobiotics via metabolic activation and/or accumulation of reactive metabolites but also might affect the clearance of other xenobiotics detoxified by the studied CYP enzymes.     

Activation of the Ahr signalling pathway by polychlorobiphenyls causes a marked induction of cytochrome P450 only after depletion of vitellogenin in Sparus aurata

The effects of polychlorinated biphenyl congeners 126 and 153 (PCB-126 and PCB-153) on vitellogenin (Vtg) and cytochrome (CYP1A1) expression were evaluated in 60 juvenile Sparus aurata. Fish were divided into four groups and the control group (Group A) was compared to fish exposed to PCB-126 (10–8 M) (Group B), PCB-153 (10–6 M) (Group C) singly and also in combination (Group D) for 12, 24 and 72 h. Hepatic expression of Vtg and CYP1A1 were analyzed using histological examinations and by immunochemical (Western blotting and immunohistochemistry) methods. Vtg increased in juvenile fishes of Groups B, C and D after 12 h respect to Group A and decreased after 24 and 72 h respect to 12 h in each group. CYP1A1 increased after 12 and 24 h in all groups vs control group and increased in Group B only at 72 h vs in control group. The results showed that chemical interaction and endocrine disruption in fish might produce deleterious consequences not only for fish but also for human.     

Pre-clinical safety assessment of the synthetic human milk,nature-identical,oligosaccharide Lacto-N-neotetraose (LNnT)

Lacto-N-neotetraose (LNnT) is a tetrasaccharide naturally occurring in human breast milk, but not in cow’s milk. The safety data generated on a potential new LNnT ingredient produced by chemical synthesis is presented. Standard in vitro genotoxicity tests were performed. LNnT was also administered via gavage in 14-, 28- and 90-day studies at levels corresponding to 0 (control), 1000, 2500 and 5000 mg/kg bw/day in juvenile rats. Fructooligosaccharide (FOS) currently approved for use in infant formulae was used as a reference control at one dose level of 5000 mg/kg bw/day. LNnT was non-mutagenic in in vitro assays. Oral administration up to 5000 mg/kg bw/day to rats over 90 days was not associated with any adverse effects, based on clinical observations, body weight gain, feed consumption, clinical pathology, organ weights and histopathology findings. Regarding gastrointestinal effects, LNnT was better tolerated than FOS during the first 2 weeks of treatment. A No Observed Adverse Effect Level (NOAEL) of 5000 mg/kg bw/day for both male and female rats was identified for LNnT when administered by gavage for 90 days. These findings in the juvenile rat support the safety of LNnT for possible use in infant foods and allow further investigation in clinical studies.     

Taiwan food scandal: The illegal use of phthalates as a clouding agent and their contribution to maternal exposure

In 2011 the Taiwan Food and Drug Administration reported that plasticizers di(2-ethylhexyl) phthalate (DEHP) and di-iso-nonyl phthalate (DiNP), endocrine disruptors, were illegally added to clouding agents used in foods and beverages. 965 products were found contaminated, of which 206 were exported to 22 countries. This study’s purpose was to obtain English names for 28 contaminated products for which DEHP levels were reported, calculate estimated average daily intake (mg/kg/day) for a 50 kg woman consuming one portion, and compare to U.S. and E.U. guidelines for daily intake. We found that drinking just one bottle (500 ml) of sports drinks would result in an average DEHP intake of 0.14 mg/kg bw/day (range 0.091–0.341), which exceeds by several fold government guidelines (0.02–0.06 mg/kg bw/day). One (2 g) serving from 4/14 samples of contaminated dietary supplements exceeds the guideline of 0.02 mg/kg bw/day. In conclusion, consuming even one portion of tainted drinks and some powders would lead to daily intake of DEHP that greatly exceeds established safety guidelines, raising concerns about potential adverse effects, particularly reproductive tract development in the male fetus. Global distribution of DEHP-contaminated and other adulterated products should prompt governments to become proactive in food safety regulations and chemical testing.     

Protective effect of Aplysin on hepatic injury in ethanol-treated rats

This study evaluated the protective effects of Aplysin against ethanol-induced hepatic injury in rats and analyzed the associated mechanisms. Rats were administered orally with ethanol 8–12 ml/kg bw excluding the rats in the control group at 1 h after rats were administered by gavage doses of Aplysin (50, 100, and 150 mg/kg bw) every day. After 6 weeks, rats were sacrificed, and liver injury was evaluated by biochemical and pathological examination. Hepatocyte apoptosis was analyzed by annexin V-FITC/PI staining. Ethanol metabolic enzymes, oxidative stress, mitochondrial function, and Bcl-2, Bax, cytochrome c and cleaved caspase-3 expressions were evaluated by western blot analysis. These results demonstrated that Aplysin exhibited a significant hepatoprotective effect. In the ethanol-treated group, cytochrome P4502E1 and alcohol dehydrogenase were increased significantly in liver tissue. Moreover, Aplysin not only significantly reversed the ratio of NAD⁺/NADH and mitochondrial glutathione depletion, but also reversed the decreased activity of mitochondrial respiratory chain complexes I, III and IV. Overexpression of cytoplasmic cytochrome c and caspase-3 activation was suppressed by Aplysin. These results suggest that Aplysin alleviates hepatocyte apoptosis by modulating the ethanol-metabolizing pathway, attenuating oxidative stress, ameliorating mitochondrial function, inhibiting mitochondrial damage-mediated apoptosis, which ultimately prevent and repair alcoholic liver injury.