Di-2-ethylhexyl phthalate (DEHP) is an environmental contaminant commonly used as a plasticizer in polyvinyl chloride products. Exposure to DEHP has been linked to adverse pregnancy outcomes in humans including preterm birth, low birth-weight, and pregnancy loss. Although oxidative stress is linked to the pathology of adverse pregnancy outcomes, effects of DEHP metabolites, including the active metabolite, mono-2-ethylhexyl phthalate (MEHP), on oxidative stress responses in placental cells have not been previously evaluated. The objective of the current study is to identify MEHP-stimulated oxidative stress responses in human placental cells. We treated a human placental cell line, HTR-8/SVneo, with MEHP and then measured reactive oxygen species (ROS) generation using the dichlorofluorescein assay, oxidized thymine with mass-spectrometry, redox-sensitive gene expression with qRT-PCR, and apoptosis using a luminescence assay for caspase 3/7 activity. Treatment of HTR-8 cells with 180 μM MEHP increased ROS generation, oxidative DNA damage, and caspase 3/7 activity, and resulted in differential expression of redox-sensitive genes. Notably, 90 and 180 μM MEHP significantly induced mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2), an enzyme important for synthesis of prostaglandins implicated in initiation of labor. The results from the present study are the first to demonstrate that MEHP stimulates oxidative stress responses in placental cells. Furthermore, the MEHP concentrations used were within an order of magnitude of the highest concentrations measured previously in human umbilical cord or maternal serum. The findings from the current study warrant future mechanistic studies of oxidative stress, apoptosis, and prostaglandins as molecular mediators of DEHP/MEHP-associated adverse pregnancy outcomes.

Introduction The dichlorofluorescein (DCF) assay is a popular method for measuring cellular reactive oxidant species (ROS). Although caveats have been reported with the DCF assay and other compounds, the potential for artifactual results due to cell-free interactions between the DCF compound and toxicants has hardly been explored. We evaluated the utility of the DCF assay for measuring ROS generation by the toxicants mono-(2-ethylhexyl) phthalate (MEHP), and tetrabromobisphenol A (TBBPA). Methods DCF fluorescence was measured spectrofluorometrically after a 1-h incubation of toxicants with 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). MEHP was incubated with carboxy-H2DCFDA in cell-free solutions of Hank’s buffered salt solution (HBSS), or in Royal Park Memorial Institute (RPMI) medium with or without fetal bovine serum. TBBPA was incubated with carboxy-H2DCFDA in cell-free HBSS and with human trophoblast cells (HTR8/SVneo cells). Results MEHP did not increase fluorescence in solutions of carboxy-H2DCFDA in HBSS or RPMI medium without serum. However, MEHP (90 and 180 μM) increased DCF fluorescence in cell-free RPMI medium containing serum. Furthermore, serum-free and cell-free HBSS solutions containing 25 μM TBBPA exhibited concentration-dependent increased fluorescence with 5–100 μM carboxy-H2DCFDA (p<0.05), but not 1 μM carboxy-H2DCFDA. In addition, we observed increased fluorescence in HTR8/SVneo cell cultures exposed to TBBPA (0.5–25 μM) (p<0.05), as we had observed in cell-free buffer. Discussion MEHP demonstrated an interaction with serum in cell-free generation of DCF fluorescence, whereas TBBPA facilitated conversion of carboxy-H2DCFDA to the fluorescent DCF moiety in the absence of serum. Because TBBPA increased fluorescence in the absence of cells, the increased DCF fluorescence observed with TBBPA in the presence of cells cannot be attributed to cellular ROS and may, instead, be the result of chemical activation of carboxy-H2DCFDA to the fluorescent DCF moiety. These data illustrate the importance of including cell-free controls when using the DCF assay to study toxicant-stimulated cellular production of ROS.

Hypocalcemia affects almost 50% of all dairy cows. Our laboratory has previously demonstrated that infusions of the serotonin precursor 5-hydroxy-l-tryptophan (5-HTP) increase circulating calcium concentrations in the Holstein transition cow. It is unknown whether feeding a negative dietary cation-anion difference (DCAD) diet alters the relationship between 5-HTP and hypocalcemia. The main objective of this study was to determine whether feeding a negative DCAD (-DCAD) diet before calving in conjunction with 5-HTP treatment could further diminish the magnitude of hypocalcemia at the time of calving. We used a randomized complete block design with a 2 × 2 factorial arrangement. Thirty-one multiparous Holstein cows were fed either a positive (+13 mEq/100 g) or negative (-13 mEq/100 g) DCAD diet 21 d before parturition and were intravenously infused daily with saline or 5-HTP (1 mg/kg) starting 7 d before the estimated date of parturition. Cows were blocked by parity and were randomly assigned to 1 of 4 treatment groups: positive DCAD plus saline, positive DCAD plus 5-HTP, negative DCAD plus saline, and negative DCAD plus 5-HTP, resulting in n = 8 per group. Total calcium (tCa), ionized calcium (iCa), and feed intake were recorded. The iCa was elevated prepartum in the -DCAD/5-HTP group compared with the other treatment groups as well as on d 0 and 1 postpartum. Although differences in tCa were not significant across the pre- or postpartum periods, tCa was numerically higher on d 0 and significantly higher on d 1 in -DCAD/5-HTP cows compared with all other groups. Prepartum the -DCAD/5-HTP treatment group ate less than the other treatment groups; however, postpartum dry matter intake differences were not significant. These findings demonstrate that feeding a -DCAD diet in conjunction with 5-HTP prepartum can increase postpartum circulating iCa concentrations and therefore diminish the magnitude of hypocalcemia at the time of parturition.

Maternal oral iron fortification mitigated PAE's disruption of fetal iron homeostasis and improved brain iron content, hematologic indices, and hepcidin production in this rat PAE model. Clinical studies show maternal ID substantially enhances fetal vulnerability to PAE, and our work supports increased maternal dietary iron intake may improve fetal iron status in alcohol-exposed pregnancies.

Maternal consumption of a high-fat diet (HFD) during pregnancy has established adverse effects on the developing neonate. In this study, we aimed to investigate the effect of an HFD on the murine mammary gland during midlactation. Female C57BL/6J mice were placed on either a low-fat diet (LFD/10% fat) or HFD (60% fat) from 3 wk of age through peak lactation (lactation day 11/L11). After 4 wk of consuming either the LFD or HFD, female mice were bred. There were no significant differences in milk yield between treatment groups, which was measured from L1 to L9. On L10, mice were subjected to an overnight fast and then euthanized on the morning of L11. Total RNA was isolated from inguinal mammary glands for whole transcriptome sequencing. We found 628 genes that were differentially expressed between the treatment groups. Notably, HFD feeding resulted in expression alterations of genes involved in collagen and cytoplasmic components. Additionally, genes related to inflammatory and immune responses were also impacted. Differential expression in gene transcript isoforms between the treatment groups was detected in three genes related to mammary duct development. This study sheds light as to how an HFD may affect the mammary gland transcriptome during midlactation.