Morphological alterations in human skeletal muscle cells exposed to di-(2-Ethylhexyl) Phthalate (dehp) in primary cell culture conditions / Alteraciones morfológicas en células musculares esqueléticas humanas expuestas a Ftalato de di-(2-etilhexilo) (dehp) en condiciones de cultivo celular primario

SUMMARY: Di-(2-ethylhexyl) phthalate (DEHP) is among the most common plasticizer additives that humans are in contact with daily. DEHP can be released from plastic and enter the human body, whereby it is metabolized and transformed into oxidative hydrophilic molecules. Clinical follow-ups in patients exposed to this phthalate and investigations in cultures of several cell types have provided information on its effects. For example, it is associated with inhibition of diploid human cell development and morphological changes in cultured germ cells. Although skeletal muscle represents around 50 % of the human body mass, knowledge about the effects of DEHP on this tissue is poor. Cultured skeletal muscle cells were exposed to DEHP (1 mM) for 13 days with the aim of exploring and evaluating some of the potential morphological effects. Three culture development parameters and nine cell characteristics were monitored during the bioassay. At 13 days, growth area, cell viability, and concentration of total proteins were lower in DEHP exposed than in control cells. Cell width and area, as well as the diameter of the nucleus and nucleolus, were greater in exposed cells than in control cells. These are interpreted as signs of cytotoxicity and suggest potential adverse effects on the development of skeletal muscle cells from DEHP exposure, as reported for other cell types.

RESUMEN: Diariamente los seres humanos tenemos contacto con aditivos plastificantes, el di-(2-etilhexil) ftalato (DEHP) se encuentra entre los más comunes. El DEHP puede liberarse del plástico e ingresar al cuerpo humano, donde es metabolizado y transformando en moléculas hidrofílicas oxidativas. Seguimientos en pacientes expuestos a este ftalato e investigaciones en cultivos de varios tipos celulares han aportado información sobre sus efectos. El DEHP es asociado con la inhibición del desarrollo de células humanas diploides y cambios morfológicos en células germinales en cultivo. Sin embargo, aún es poco lo que se sabe sobre los efectos en el músculo esquelético, a pesar de que este tejido representa alrededor del 50 % de la masa corporal del humano. Para explorar y evaluar algunos efectos morfológicos en células de músculo esquelético, cultivos primarios fueron expuestos a DEHP (1 mM) durante 13 días. Se dio seguimiento a tres parámetros de desarrollo del cultivo y nueve características celulares. Al término de 13 días de exposición, los valores del área de crecimiento, viabilidad celular y concentración de proteínas totales fueron inferiores con respecto a los cultivos control. Se observaron cambios morfométricos en las células expuestas. Particularmente, el ancho y área celular, así como los diámetros del núcleo y nucleolos, fueron mayores a los registros en las células control. Estos resultados se interpretan como signos de citotoxicidad y sugieren efectos potencialmente adversos en el desarrollo de las células del músculo esquelético ante una exposición al DEHP, como se ha registrado para otros tipos celulares.

Differential toxicological endpoints of di(2-ethylhexyl) phthalate (DEHP) exposure in MCF-7 and MDA-MB-231 cell lines: Possible estrogen receptor α (ERα) independent modulations.

Wide spread use of Di-(2-ethylhexyl) phthalate (DEHP) has made it a ubiquitous contaminant in today’s environment, responsible for possible carcinogenic and endocrine disrupting effects. In the present investigation an integrative toxico-proteomic approach was made to study the estrogenic potential of DEHP. In vitro experiments carried out with DEHP (0.1-100 μM) induced proliferations (E-screen assay) in human estrogen receptors-α (ERα) positive MCF-7 and ERα negative MDA-MB-231 breast cancer cells irrespective of their ERα status. Further, DEHP suppressed tamoxifen (a potent anti-breast cancer drug) induced apoptosis in both cell types as shown by flowcytometric cell cycle analysis. Label-free quantitative proteomics analysis of the cell secretome of both the cell lines indicated a wide array of stress related, structural and receptor binding proteins that were affected due to DEHP exposure. The secretome of DEHP treated MCF-7 cells revealed the down regulation of lactotransferrin, an ERα responsive iron transport protein. The results indicated that toxicological effects of DEHP did not follow an ERα signaling pathway. However, the differential effects in MCF-7 and MDA-MB-231 cell lines indicate that ERα might have an indirect modulating effect on DEHP induced toxicity.

Disruption of reproductive development in male rat offspring following gestational and lactational exposure to di-(2-ethylhexyl) phthalate and genistein

Studies of developmental effects of mixtures of endocrine disrupters on the male reproductive system are of great concern. In this study, the reproductive effects of the co-administration of di-2-(ethylhexyl) phthalate (DEHP) and genistein (GEN) during pregnancy and lactation were studied in male rat offspring. Pregnant Sprague-Dawley rats were gavaged from gestation day 3 to postnatal day 21 with vehicle control, DEHP 250 mg/kg body weight (bwyday, GEN 50 mg/kg bwday, GEN 400 mg/kg bwday, and two combinations of the two compounds (DEHP 250 mg/kg bwday + GEN 50 mg/kg bwday, DEHP 250 mg/kg bwday + GEN 400 mg/kg bwday). The outcomes studied were general morphometry (weight, AGD), testicular histology, testosterone levels, and expression at the mRNA level of genes involved in steroidogenesis. Organ coefficient, AGD / body weight1/3 י, serum testosterone concentration and genes involved in steroidogenic pathway expression when exposed to DEHP (250mg/kg bwday), GEN(50mg/kg bwday) or GEN(400mg/kg bwday) alone were not significantly different from the control group. When exposed to (DEHP 250mg/kg bwday +GEN 50mg/kg bwday) together during pregnancy and lactation, serum testosterone concentration, epididymis coefficient and Cypal17a1,Scarb1 m RNA expression significantly decreased compared to the control and GEN(50mg/kg bwday). When exposed to (DEHP 250mg/kg bwday +GEN 400mg/kg bwday) together during pregnancy and lactation, AGD / body weight1/3 י, serum testosterone concentration, testis and epididymis coefficient and Star, Cypal17a1 mRNA expression appeared significantly decreased compared to the control and DEHP/GEN single exposure, together with developmental impairment of seminiferous tubules and seminiferous epithelium. Overall, co-administration of DEHP and GEN during gestation and lactation seem to acts in a cumulative manner to induce the most significant alterations in the neonate, especially with GEN at high dose, although the effect of the DEHP-GEN mixture on adult offspring should be observed further.

[ Effect of Di-[2-Ethylhexyl] Phthalate [DEHP] on Mouse Daily Sperm Production and Epididymal Sperm Parameters]

The purpose of tins study is to evaluate the effect of Di-[2-ethylhexyl] phthalate [DEHP] ontesticular spermatid number per gram testis [TSN]. daily sperm production [DSP], count, motility, viability morphology, and chromatine quality of epididymal sperm. The protocol for DEHP administration was [hat adult male NMRI mice [the age group or 4 weeks] received 2g DEHP/100ul corn oil/kg, and vehicle group received l00uI corn oil/kg by gavage for 14 days. The control group did not receive DEHP. All the salliples were assessed according to World Health Organization [WHO] criteria. Sperm morphology was assessed using papanicula staining Sperm chromatine quality was assessed using aniline-blue staning Results: Administration of DEHP induced significantly reduction of TSN, DSP and epididymal sperm count [p < 0.05]. The percentage of motility, viability, normal morphology, and chromosomal quality were significantly low in comparison with control group [p < 0.05]. These results demonstrated that DEHP administration has toxic effects on TSN, DEP. Epididymal sperm parameter and finally on male reproductive system

Reduced viability of F1 egg ropes in Chironomus riparius exposed to di-2-ethylhexyl phthalate (DEHP).

The midge (Chironomus riparius), an aquatic invertebrate, was exposed to di-2-ethylhexyl phthalate (DEHP) to evaluate the effects on reproductive processes via sediment toxicity. The test endpoints included emergence, sex ratio, fecundity and viability of F1 offspring egg ropes. After exposures of C. riparius to DEHP in this study, a significant result was observed only in the viability test of F1 egg ropes. That is, hatching rates of F1 egg ropes from DEHP exposed adults were decreased significantly. This result provides the evidence that DEHP could lead abnormal egg ropes development by DEHP exposure during larval period. This means that DEHP could effect to Chironomus population density through reduced F1 offspring.

Administration of the plasticizer di (2-ethylhexyl) phthalate alters glycoconjugate profile.

This study was undertaken to evaluate the level of glycoproteins and sialic acid in rats fed di (2-ethylhexyl)-phthalate (DEHP) in the diet for 24 weeks. Protein-bound hexose, hexosamine and sialic acid were increased in plasma and liver of rats treated with DEHP, whereas the erythrocyte membrane showed a reduction following DEHP administration. Evaluation of glycoproteins is a useful indicator of the carcinogenic process. It is suggested that profound alterations in membrane components observed in the present study may be related to the carcinogenic potential of DEHP.

Effect of in utero exposure to di(2-ethylhexyl)phthalate on rat testes.

In utero exposure to di(2-ethylhexyl)phthalate (DEHP; 1000 mg/kg body weight) significantly decreased activities of testicular sorbitol dehydrogenase and acid phosphatase and increased gamma-glutamyl transpeptidase, lactate dehydrogenase and beta-glucuronidase activities at early ages. A decrease in the sperm count of the epididymal spermatozoa was also observed in the sexually matured animals of DEHP exposed group. The data suggest that in utero exposure to DEHP may affect the normal development of testes.

Biochemical alterations in rat fetal liver following in utero exposure to di(2-ethylhexyl)phthalate (DEHP).

Oral administration of DEHP, 1000 mg/kg body weight, to rats daily from 6 to 15 day of gestation resulted in retardation of fetal growth and increase in fetal liver weight which contained significant quantities of DEHP. The activities of mitochondrial succinate dehydrogenase, malate dehydrogenase, cytochrome c oxidase and adenosine triphosphatase were decreased in fetal liver. The data indicate that exposure of mothers to DEHP during pregnancy could adversely affect the fetal livers by interfering with bioenergetics of the cell.