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Although Mono (2-ethylhexyl) phthalate (MEHP) is a metabolite of Di (2-ethylhexyl) phthalate (DEHP), it has been confirmed to exhibit stronger biological toxicity than DEHP. Mitochondrial dynamic homeostasis and normal mitochondrial function regulate numerous physiological and pathological processes. However, it remains unclear whether MEHP triggers apoptosis, autophagy, and cell cycle arrest in grass carp kidney (CIK) cells by causing mitochondrial damage. Here, we established a MEHP dose-dependent exposure models in CIK cells and treated them with NAC. The results demonstrated that MEHP promoted ROS production and decreased antioxidant enzyme activities in CIK cells in a concentration-dependent manner. MEHP destroyed mitochondrial homeostasis and mitochondrial function in CIK cells, manifested by decreasing mitochondrial membrane potential (MMP), down-regulating gene expression of fusion division genes including MFN1, MFN2, CLPP, DRP1, OPA1, and MFF, and reducing OXPHOS complex enzyme protein level including COXI, COXII, COXIII, COXIV, and COXV. In addition, MEHP treatment not only can increase the level of Cyt-c, Atg12, Atg13, Atg14, Beclin1, ULK1, LC3-II, Caspase3, Caspase9, and Bax, but also can decrease the level of Bcl2, p62, CyclinB, CyclinD, and CyclinE in a concentration-dependent manner, which resulted in apoptosis, autophagy and cell cycle arrest. Furthermore, MEHP dose-dependently nduced downregulation gene expression of immunoglobulins and antimicrobial peptides (Hepcidin, β-defensin, LEAP2). However, NAC treatment could significantly reverse the above changes and alleviate CIK cells damage caused by exposure to MEHP. This study has expanded our understanding about molecular mechanisms of MEHP toxicity in aquatic animals and provided a reference for comparative medicine research., Competing Interests: Declaration of competing interest The authors have announced no conflict of interest. All authors have read the manuscript and consented to submit it in its current form for consideration for publication in the Journal., (Copyright © 2024. Published by Elsevier Inc.)

Combined exposure to phthalate esters (PAEs) has garnered increasing attention due to potential synergistic effects on human health. This study aimed to develop an in vitro model using human macrophages to evaluate the combined toxicity of PAEs and explore the underlying mechanisms. A high-throughput screening system was engineered by expressing a PPRE-eGFP reporter in THP-1 monocytes to monitor macrophage polarization upon PAEs exposure. Individual PAEs exhibited varied inhibitory effects on M2 macrophage polarization, with mono(2-ethylhexyl) phthalate (MEHP) being the most potent. Isobologram analysis revealed additive interactions when MEHP was combined with other PAEs, resulting in more pronounced suppression of M2 markers compared to individual compounds. Mechanistic studies suggested PAEs may exert effects by modulating PPARγ activity to inhibit M2 polarization. Notably, an equimolar mixture of six PAEs showed additive inhibition of M2 markers. In vivo experiments corroborated the combined hepatotoxic effects, with mice exposed to a PAEs mixture exhibiting reduced liver weight, dyslipidemia, and decreased hepatic M2 macrophages compared to DEHP alone. Transcriptome analysis highlighted disruptions in PPAR signaling, and distinct pathway alterations on cholesterol metabolism in the mixture group. Collectively, these findings underscore the importance of evaluating mixture effects and provide a novel approach for hazard assessment of combined PAEs exposure with implications for environmental health risk assessment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Di(2-ethylhexyl) phthalate (DEHP) is a chemical widely distributed in the environment as is extensively used in the plastic industry. DEHP is considered an endocrine disruptor chemical (EDC) and humans are inevitably and unintentionally exposed to this EDC through several sources including food, beverages, cosmetics, medical devices, among others. DEHP exposure has been associated and may be involved in the development of various pathologies; importantly, pregnant women are a particular risk group considering that endocrine alterations during gestation may impact fetal programming leading to the development of several chronic diseases in adulthood. Recent studies have indicated that exposure to DEHP and its metabolite Mono(2-ethylhexyl) phthalate (MEHP) may impair placental development and function, which in turn would have a negative impact on fetal growth. Studies performed in several trophoblastic and placental models have shown the negative impact of DEHP and MEHP in key processes related to placental development such as implantation, differentiation, invasion and angiogenesis. In addition, many alterations in placental functions like hormone signaling, metabolism, transfer of nutrients, immunomodulation and oxidative stress response have been reported. Moreover, clinical-epidemiological evidence supports the association between DEHP exposure and adverse pregnancy outcomes and pathologies. In this review, we aim to summarize for the first time current knowledge about the impact of DEHP and MEHP exposure on placental development and pathophysiology, as well as the mechanisms involved. We also remark the importance of exploring DEHP and MEHP effects in different trophoblast cell populations and discuss new perspectives regarding this topic., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The purpose of the present study was to examine the antioxidant and oxidative stress changes in zebrafish liver (ZFL) cells in the presence of mono-(2-ethylhexyl) phthalate (MEHP). When reactive oxygen species (ROS) and antioxidant levels were measured by immunoassay, significant differences were observed between MEHP-treated and control cells, while catalase levels did not change in any group. MEHP-treated cells had higher levels of ROS, glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione, and superoxide dismutase (SOD) than control cells. However, lower levels of lipid peroxidation were observed in MEHP-treated cells compared to control cells. After 24 h of MEHP treatment, ROS, SOD, GPx, and GST activity increased in a dose-dependent manner. Cellular lipid droplet formation and endoplasmic reticulum stress were both induced in the presence of MEHP. These findings demonstrated the potential impacts of the association of MEHP with adverse outcomes in fish liver. Future studies will focus on clarifying the molecular mechanism of phthalate toxicity via oxidative stress and peroxisome proliferator activated receptor as the major mechanistic pathway., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Histiotrophic nutrition pathways (HNPs) are processes by which the organogenesis-stage conceptus obtains nutrients, amino acids, vitamins and cofactors required for protein biosynthesis and metabolic activities. Nutrients are captured from the maternal milieu as whole proteins and cargoes via receptor-mediated endocytosis in the visceral yolk sac (VYS), degraded by lysosomal proteolysis and delivered to the developing embryo (EMB). Several nutrients obtained by HNPs are required substrates for one-carbon (C1) metabolism and supply methyl groups required for epigenetic processes, including DNA and histone methylation. Increased availability of methyl donors has been associated with reduced risk for neural tube defects (NTDs). Here, we show that mono-2-ethylhexyl phthalate (MEHP) treatment (100 or 250μM) alters HNPs, C1 metabolism and epigenetic programming in the organogenesis-stage conceptus. Specifically, 3-h MEHP treatment of mouse EMBs in whole culture resulted in dose-dependent reduction of HNP activity in the conceptus. To observe nutrient consequences of decreased HNP function, C1 components and substrates and epigenetic outcomes were quantified at 24h. Treatment with 100-μM MEHP resulted in decreased dietary methyl donor concentrations, while treatment with 100- or 250-μM MEHP resulted in dose-dependent elevated C1 products and substrates. In MEHP-treated EMBs with NTDs, H3K4 methylation was significantly increased, while no effects were seen in treated VYS. DNA methylation was reduced in MEHP-treated EMB with and without NTDs. This research suggests that environmental toxicants such as MEHP decrease embryonic nutrition in a time-dependent manner and that epigenetic consequences of HNP disruption may be exacerbated in EMB with NTDs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Phthalates are suspected of having adverse effects on androgen-regulated reproductive development in animals and may be toxic for human sperm. The purposes of our study were to investigate the general exposure of a Chinese reproductive age cohort to these ubiquitous pollutants and to assess their potential effect on semen quality. Six phthalate metabolites, monomethyl phthalate (MMP), monoethyl phthalate (MEP), monobutyl phthalate (MBP), monobenzyl phthalate (MBzP), mono-2-ethylhexyl phthalate (MEHP), and mono-2-ethyl-5-oxohexyl phthalate (MEOHP) were measured in spot urines of 150 individuals recruited from a Chongqing, China, reproductive institute. The questionnaire and clinical data were evaluated, and the correlations of phthalate exposure and semen qualities like semen volume, sperm concentration, motility and sperm motion parameters, were determined by multiple logistic regression analysis. The creatinine adjusted average concentrations for MMP, MEP, MBP, MBzP, MEHP and MEOHP were 41.3, 300, 41.0, 0.78, 2.99 and 3.90 μg/g, respectively. After adjustment for age, body mass index (BMI), abstinence, smoking, drinking, and education, there was a borderline-significant dose-response relationship between MBP and sperm concentration, with odd ratios (ORs) 1.0, 6.8 and 12.0 for increasing exposure tertiles (p=0.05). Although the dose-response relationships for MMP and MEP versus sperm concentration were not significant, a significant positive correlation between MEP and straight-line velocity of sperm motion was observed. The present data may imply some effects of phthalate exposure on semen. However, due to the small sample size, our finding needs to be confirmed on a larger population., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Sources of phthalates other than Polyvinyl chloride (PVC) related products are scarcely documented in Mexico. The objective of our study was to explore the association between urinary levels of nine phthalate metabolites and the use of personal care products. Subjects included 108 women who participated as controls in an ongoing population-based case-control study of environmental factors and genetic susceptibility to breast cancer in northern Mexico. Direct interviews were performed to inquire about sociodemographic characteristics, reproductive history, use of personal care products, and diet. Phthalate metabolites measured in urine by high performance liquid chromatography-isotope dilution tandem mass spectrometry were monoethyl phthalate (MEP), monobenzyl phthalate (MBzP), mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), mono-3-carboxypropyl phthalate (MCPP) as well as mono-2-ethylhexyl phthalate (MEHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP), mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP), mono-2-ethyl-5-carboxypentyl phthalate (MECPP) that are metabolites of di-ethylhexyl phthalate (DEHP). Detectable urinary concentrations of phthalate metabolites varied from 75% (MEHP) to 100% (MEP, MBP, MEOHP, MEHHP and MECPP). Medians of urinary concentrations of some phthalate metabolites were significantly higher among users of the following personal care products compared to nonusers: body lotion (MEHHP, MECPP and sum of DEHP metabolites (ΣDEHP)), deodorant (MEHP and ΣDEHP), perfume (MiBP), anti-aging facial cream (MEP, MBP and MCPP) and bottled water (MCPP, MEHHP and MEOHP). Urinary concentrations of MEP showed a positive relationship with the number of personal care products used. Our results suggest that the use of some personal care products contributes to phthalate body burden that deserves attention due to its potential health impact., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Selenium is an essential cofactor in the key enzymes involved in cellular antioxidant defense. It plays a critical role in testis and reproduction and regulates DNA damage within the prostate. Phthalates are ubiquitous environmental contaminants that cause alterations in endocrine and spermatogenic functions in animals. The objective of this study was to investigate the cytotoxicity and genotoxicity potentials of di(2-ethylhexyl)phthalate (DEHP), the most widely used phthalate and its primary toxic metabolite mono(2-ethylhexyl)phthalate (MEHP), and their effects on the antioxidant balance in the LNCaP human prostate adenocarcinoma cell line. Protection by selenium supplementation with either sodium selenite (SS, 30 nM) or selenomethionine (SM, 10 microM) was also investigated. Both DEHP (3mM) and MEHP (3 microM) caused significant decreases in cell viability; altered antioxidant status, particularly decreasing the GPx1 activity; and induced DNA damage as measured by the alkaline comet assay. Selenium supplementation was highly protective against cytotoxicity, partially prevented genotoxicity, and restored the antioxidant status. The results of this study suggested that the underlying mechanism of cytotoxicity and resulting disturbances produced by DEHP or MEHP was an an oxidative stress process and/or an effect on the expression of antioxidant enzymes, and accentuated the importance of selenium status, particularly with respect to the high probability of phthalate exposures and their adverse effects., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The risk assessment of di(2-ethylhexyl)phthalate (DEHP) migrating from polyvinyl chloride (PVC) medical devices is an important issue. Many studies have been conducted to determine the level of DEHP migration. A recent report has indicated that DEHP in blood bags is hydrolyzed by esterase into mono(2-ethylhexyl)phthalate (MEHP). However, MEHP is thought to be even more toxic than the parent compound. Therefore, a method for the simultaneous determination of DEHP and MEHP was developed. The limits of quantification (LOQs) of DEHP and MEHP were 2.5 and 0.75 ng/ml, respectively. In this study, the effect of sterilization process on the levels of DEHP and MEHP migration was investigated. The level of migration of DEHP from gamma(gamma)-ray sterilized PVC sheet was low compared with that of the unsterilized control. By contrast, the level of MEHP migration from the gamma-ray sterilized PVC sheet was high compared with that of the unsterilized control. In addition, a high content of MEHP was found in the gamma-ray sterilized PVC sheet.

Phthalates are used primarily as plasticizers to make polyvinyl chloride (PVC) soft and flexible. In recent years the phthalate esters have attracted increasing attention as environmental and biomedical pollutants and, because of their toxicological characteristics. In particular, they are more and more recognized as endocrine disrupters. In this context, we describe herein an efficient synthetic pathway leading to a series of metabolites of di(2-ethylhexyl) phthalate (DEHP). Mono(2-ethylhexenyl) phthalate was used as starting material to obtain these products in good yield, large scale and GC-MS purity. The metabolites of DEHP were synthesized, for the first time, as biomarkers to verify their quantitative determination in human urine and serum by GC-MS analysis for studying the exposure to phthalates and establishing reference values.

Primary hepatocyte cultures derived from rat, rabbit, guinea pig and monkey have been treated in vitro with metabolites of di(2-ethylhexyl)phthalate, i.e. mono(2-ethylhexyl)phthalate (MEHP), mono(5-carboxy-2-ethylpentyl)phthalate (metabolite V) and mono(2-ethyl-5-oxohexyl)phthalate (metabolite VI). In rat hepatocyte cultures MEHP and metabolite VI were equally potent in inducing peroxisome proliferation, while metabolite V was much less potent. In rat hepatocytes a 50% increase in both peroxisomal palmitoyl-CoA oxidase activity and microsomal lauric acid omega-hydroxylation activity was found after treatment with 5-15 microM MEHP. In guinea pig, rabbit and monkey hepatocyte cultures, a 50% increase in peroxisomal palmitoyl-CoA oxidase activity was found after treatment with 408-485 microM MEHP. No induction of lauric acid omega-hydroxylation activity was found. These results indicate that peroxisome proliferation can be induced by MEHP in rabbit, guinea pig and monkey hepatocytes, but that these species are at least 30-fold less sensitive to peroxisome proliferation induction than rats. The proposed mechanistic inter-relationship between induction of lauric acid omega-hydroxylation activity and peroxisome proliferation is found in rat hepatocytes, but not in hepatocytes of the other three species. Treatment of guinea pig hepatocyte cultures with MEHP resulted in an increase in triglyceride concentrations in the hepatocytes. In rat and rabbit hepatocyte cultures, triglyceride concentrations were much less altered by MEHP. In monkey hepatocytes a decrease in hepatic triglyceride concentration was found after treatment with MEHP. These effects are in agreement with in vivo effects observed before. After treatment of primary hepatocyte cultures with MEHP, high concentrations of omega- and (omega-1)-hydroxylated metabolites of MEHP were found in media from rat, rabbit and guinea pig cultures. The formation of these metabolites did not decline in time. During treatment the metabolite profile in media from rat hepatocyte cultures moved towards omega-hydroxy metabolites of MEHP. In media from monkey hepatocyte cultures the lowest concentrations of hydroxylated metabolites were determined. No major species differences were found in the potency to form oxidized MEHP metabolites, and thus no unique metabolite differences were found, which could explain the species differences in sensitivity for peroxisome proliferation.

In order to investigate a proposed relationship between induction of hepatic microsomal lauric acid hydroxylase activity and peroxisome proliferation in the liver, male Wistar rats were treated with peroxisome proliferating compounds, and the lauric acid hydroxylase activity, the immunochemical detectable levels of cytochrome P450 4A1 and the activities of peroxisomal enzymes were determined. In addition, the levels of cytochrome P450 4A1 and lauric acid hydroxylase activities were studied after treatment of rats with three cytochrome P450 inducers. After treatment with aroclor-1254, phenobarbital or 3-methylcholanthrene total cytochrome P450 was 1.7-2.7 times induced. However, no induction of lauric acid omega-hydroxylase activities or P450 4A1 levels were found. After treatment of rats with di(2-ethylhexyl)phthalate (DEHP) a dose-dependent induction of lauric acid omega-hydroxylase activities, levels of cytochrome P450 4A1 and peroxisomal fatty acid beta-oxidation was found. Even at a dose-level of 100 mg DEPH/kg body weight per day a significant induction of these activities was observed. The main metabolites of DEHP, mono(2-ethylhexyl)phthalate and 2-ethyl-1-hexanol, also caused an induction of levels of P450 4A1, lauric acid omega-hydroxylase activities and the activity of peroxisomal palmitoyl-CoA oxidase. 2-Ethyl-1-hexanoic acid did not influence lauric acid omega-hydroxylase activities, but did induce levels of P450 4A1 and palmitoyl-CoA oxidase activities. Three other compounds (perfluoro-octanoic acid, valproate and nafenopin) induced both lauric acid omega-hydroxylase activity and peroxisomal palmitoyl-CoA oxidase activity. The plasticizer, di(2-ethylhexyl)adipate, did not induce levels of P450 4A1, lauric acid omega-hydroxylase activities or palmitoyl-CoA oxidase activities. With the compounds tested a close association between the induction of lauric acid omega-hydroxylase activities and peroxisomal palmitoyl-CoA oxidase activity was found. These data support the theory that peroxisome proliferating compounds do induce lauric acid omega-hydroxylase activities and that there might be a mechanistic inter-relationship between peroxisome proliferation and induction of lauric acid omega-hydroxylase activities.

Monoethylhexyl phthalate, at concentrations that can occur in blood stored in plastic bags (0.1-0.5 mg/ml), reduced contractions of rat isolated gastric fundus to PGE2 and acetylcholine; the diethyl compound was less effective. In contrast, dibutyl phthalate (1 and 10 micrograms/ml) and, to a lesser extent di-isobutyl phthalate, increased the muscle tone. These results are discussed in relation to blood transfusion, and to structural similarities between phthalates and prostaglandins.

In cultured hepatocytes, as in vivo, mono-2-ethylhexyl phthalate (MEHP) and its straight chain analogues mono-n-hexyl phthalate (MnHP) and mono-n-octyl phthalate (MnOP) each cause accumulation of lipid but only MEHP produces significant induction of peroxisomal fatty acid oxidizing enzymes. To elucidate the mechanisms underlying this lipid accumulation we investigated the effects of these phthalates and the drug clofibric acid on fatty acid metabolism in suspensions of isolated hepatocytes. The effects were found to be markedly dependent on the nutritional state of the animals from which the hepatocytes were isolated. In hepatocytes isolated from animals fasted overnight, or animals fed ab libitum but killed at approximately 2.30 p.m., MEHP, MnHP, MnOP and clofibric acid each caused a marked rapid stimulation of fatty acid oxidation and the synthesis of triglycerides in hepatocytes when incubated in Hanks saline. Export of very low density lipoprotein (VLDL) from the cells was either unchanged or somewhat reduced. In contrast, in hepatocytes isolated from rats fed ad libitum but killed at approximately 9.30 a.m. MEHP and clofibric acid did not alter fatty acid oxidation or triglyceride synthesis, while MnOP and MnHP increased triglyceride synthesis but decreased fatty acid oxidation. The effects of fasting were largely abolished by incubations of the cells in a complete tissue culture medium (Liebowitz L-15). The results suggest that MEHP and its straight chain analogues can, either as the free acid or the CoA ester, mimic the action of fatty acids in the allosteric regulation of fatty acid metabolism.

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.