Zinc status alters growth and oxidative stress responses in rat hepatoma cells.

Zinc deficiency and excess influence cellular homeostasis and are believed to modulate apoptosis. Zinc also regulates cell growth and proliferation. Understanding of the role of zinc in the mechanisms associated with these changes is limited because of its diverse, complex, and cell-specific effects. Therefore, we investigated the oxidative stress responses and the underlying molecular mechanisms associated with the disruption of intracellular zinc homeostasis in H4IIE rat hepatoma cells. We found that zinc excess (100 µM) and DTPA (diethylenetriaminepentaacetic acid; 50-100 µM) induced zinc deficiency both generate reactive oxygen species (ROS) and decrease viability in H4IIE cells. However, cotreatment with the antioxidant, N-acetyl-L-cysteine (NAC) both reduced ROS production and protected cells from death. We additionally observed an increase in Bax mRNA and cytochrome c release from the mitochondria in DTPA-treated cells and an elevated expression of Fas/Fas ligand mRNA with zinc treatment. Both treatments increased p53 and MdM2 protein concentrations along with caspase 3/7 activity. These results suggest that zinc deficiency stimulates mitochondrial-dependent apoptosis whereas zinc activates the extrinsic-apoptotic pathway. Both decreasing and increasing cellular zinc concentrations modulate ROS mediated apoptosis and warrant further research on zinc mediated cancer chemoprevention in this and other cancer cell lines.

24-Hour Dietary Recall in the Expanded Food and Nutrition Education Program: Perspective of the Program Coordinator.

The purpose of this study was to determine how the 24-hour dietary recall (24HDR) is administered and how the Expanded Food and Nutrition Education Program (EFNEP) peer educators and other staff are trained on the data collection and entry process, from the EFNEP coordinators' perspectives. This cross-sectional, quantitative study utilized an online survey to collect information from EFNEP coordinators representing 61 of 76 EFNEP programs. While 56% of the programs collected the 24HDR data starting with the first thing eaten the previous day, 49% of them started collecting data at the time of class, going backwards. Most programs, i.e., 72%, reported using a multiple-pass method; however, only one-third of them reported using the standard five-pass method. Almost all programs, i.e., 97%, reported one peer educator collecting data from a group of 2-12 clients. All programs reported collecting the 24HDR data in a group setting, with about one-third of the programs also collecting data one-on-one. Most programs, i.e., 57%, reported spending ≤4 h on the initial training of staff in how to collect 24HDR data, and 54% of them reported that the peer educators entered the data themselves. This study found that the methods used to collect answers, train the staff, and enter the 24HDR data varied across EFNEP programs and that there is a need to standardize or revise the collection of 24HDR data.

Conducting 24-Hour Dietary Recalls in Group Settings with Adults Having Low-Income: Perspectives of EFNEP Peer Educators.

The Expanded Food and Nutrition Education Program (EFNEP) is a federally funded program that teaches nutrition education to adults and youth with low-income. EFNEP is funded throughout the United States including federal territories. The purpose of EFNEP is to provide nutrition education. Evaluation for adult programs includes pre/post surveys and pre/post 24-h diet recalls (24HDR). A validated standard of dietary measures, 24HDR are useful when collected as designed: one-on-one by a trained professional. In EFNEP, 24HDR are collected in group settings by EFNEP peer educators who often have not received a college degree or any formal education in nutrition. The purpose of this study was to explore attitudes and behaviors of EFNEP peer educators regarding how they collect diet recalls in a group setting, their perceptions of how adult participants feel about the recalls, and the benefits and challenges of using recalls. Online interviews were conducted with EFNEP peer educators across the U.S. Peer educators recognized the importance of collecting the recall data but identified several challenges such as time, resources, and participant reluctance to complete the recall. Program evaluation through methods like the 24HDR is important to measure outcomes and inform program improvements but also needs to include how evaluation can benefit participants and minimize data collection burden. Future research needs to examine the validity of collecting recalls in a group setting compared to other measures of diet quality.

Diet Quality and Nutrition Behavior of Federal Nutrition Education Program Participants before and during the COVID-19 Pandemic.

Despite challenges due to the COVID-19 pandemic, reports from regional and national meetings of the Expanded Food and Nutrition Education program (EFNEP) have provided anecdotal evidence that the program has persevered, pivoted, and continued to positively impact the lives of some of the nation's most vulnerable populations. However, there have been necessary changes to program delivery, inevitable changes in the lives of participants, and changes in the food environment that may have impacted program outcomes. This study compares national EFNEP data (demographics, behavior change data, and 24 h dietary recall data) of participants from two federal fiscal years, before the COVID-19 pandemic and during the pandemic. Linear mixed model analysis of variance and covariance were used to assess the effects of year on program outcomes. Results of this study provide quantitative evidence of the resiliency of EFNEP to facilitate positive behavior changes related to diet quality, physical activity, food safety, food resource management, and food security. Amidst changes in the food environment during the COVID-19 pandemic, these results emphasize the importance and value of federal nutrition education programs in any food environment.

The Relationship between Macronutrient Distribution and Type 2 Diabetes in Asian Indians.

Asian Indians (AIs) are at increased risk for type 2 diabetes mellitus than other ethnic groups. AIs also have lower body mass index (BMI) values than other populations, so can benefit from strategies other than weight reduction. Macronutrient distributions are associated with improved glycemic control; however, no specific distribution is generally recommended. This study looks at whether a macronutrient distribution of 50:30:20 (percent of total calories from carbohydrates, fats, and protein) is related to diabetes status in AIs. Diet and Hemoglobin A1c (HbA1c) were assessed from convenience sample of AI adults in Maryland. A ratio of actual to needed calories using the 50:30:20 macronutrient distribution was then tested against diabetes status to identify associations. All groups except non-diabetic females, were in negative energy balance. The non-diabetic group consumed larger actual to needed ratios of protein than pre-diabetics and diabetics. However, all groups consumed protein at the lower end of the Acceptable Macronutrient Distribution Range (AMDR), and the quality of all macronutrients consumed was low. Therefore, weight loss may not be the recommendation for diabetes management for AIs. Increasing protein and insoluble fiber consumption, could play a critical role.

The effects of transformation and ZnT-1 silencing on zinc homeostasis in cultured cells.

We have previously demonstrated that reducing the availability of zinc with the extracellular chelator diethylenetriaminepentaacetic acid (DTPA) promotes efflux of (65)Zn from rat primary hepatocytes and pituitary cells, but increases retention of label in rat hepatoma (H4IIE) and anterior pituitary tumor (GH3) cell lines. To further understand this differential response between primary cells and the corresponding cancer cell lines, we investigated the effects of immortalizing primary cells on their zinc homeostasis. Rat primary hepatocytes were electroporated with the SV40 large T-antigen-coding plasmid pSV3-neo and selected for neomycin resistance. This resulted in cell division of the normally quiescent hepatocytes. When these cells were prelabeled with (65)Zn, DTPA decreased efflux of (65)Zn, similarly to hepatoma cells and differently from primary hepatocytes. This homeostatic change may be required to account for the greater zinc requirements of dividing cells and be mediated by alterations in the activity of zinc transporter ZnT-1, which is responsible for zinc efflux. To further understand the mechanism of DTPA-induced zinc retention, we down-regulated the expression of ZnT-1 in rat hepatoma cells using vector-based short hairpin RNA interference. Expression of ZnT-1 protein was reduced to approximately 50%. Down-regulation of ZnT-1 resulted in greater retention of (65)Zn in control cells. However, DTPA increased rather than decreased efflux of label from knockdown cells, suggesting that functional ZnT-1 is required for the decreased efflux in response to DTPA. We conclude that ZnT-1 expression is crucial for maintaining zinc homeostasis, in particular, for the enhanced retention of zinc in transformed cells when subjected to zinc deprivation.

Rapid homeostatic response of H4IIE cells to diethylenetriaminepentaacetic acid is not due to changes in the amount or localization of ZnT-1 protein.

We have demonstrated that reducing zinc availability with the extracellular chelator diethylenetriaminepentaacetic acid (DTPA) causes rapid inhibition of cellular zinc efflux in H4IIE hepatoma cells but increases zinc efflux in primary hepatocytes. Similar differences were also observed between the rat anterior pituitary cell line GH3 and primary anterior pituitary cells. We hypothesized that the difference between the transformed and primary cells is due to differential regulation of ZnT-1 or SLC-30A-1 because this is the only zinc efflux transporter localized to the plasma membrane. The effects of DTPA (50 µM) and zinc (100 µM) treatment on messenger RNA (mRNA) and protein expression and protein localization of ZnT-1 were studied in H4IIE cells and primary hepatocytes. Although zinc tended to increase ZnT-1 mRNA in H4IIE cells, DTPA had no effect on ZnT-1 mRNA and protein expression in either hepatoma cells or hepatocytes. Although ZnT-1 is thought to be localized on the plasma membrane, this localization was not seen in these liver cells where ZnT-1 was distributed throughout the cytoplasm. Vesicular localization of ZnT-1 appeared to increase with zinc treatment. Total zinc content was reduced by DTPA in H4IIE cells. Diethylenetriaminepentaacetic acid also reduced metallothionein 1 mRNA, reflecting this reduction in cellular zinc. We conclude that the rapid homeostatic response of cells to altered zinc availability must be attributed to a transporter other than ZnT-1 or to changes in the activity of ZnT-1 by a novel mechanism.

Zinc retention differs between primary and transformed cells in response to zinc deprivation.

Previous studies in our laboratory have demonstrated that reducing the availability of zinc with the extracellular metal chelator DTPA (diethylenetriaminepentaacetate) enhances, rather than inhibits, the thyroid hormone induction of growth hormone mRNA in GH3 rat anterior pituitary tumor cells. To understand the actions of the chelator on cellular zinc status, we observed the effects of DTPA on (65)Zn uptake and retention. DTPA reduced the uptake of (65)Zn by GH3 cells from the medium, but when GH3 cells were prelabeled with (65)Zn, it resulted in greater retention of the isotope. In primary hepatocytes, DTPA both reduced the uptake of (65)Zn from the medium and increased efflux from prelabeled cells. To investigate this difference, we studied the effects of DTPA on radioactive zinc flux in the H4IIE (rat hepatoma), MCF-7 (human breast cancer) and Hs578Bst (nontransformed human mammary) cell lines and in rat primary anterior pituitary cells. DTPA reduced the uptake of (65)Zn in all cell lines examined. DTPA increased the retention of (65)Zn in prelabeled H4IIE, MCF-7 and Hs578Bst cells but reduced it in primary pituitary cells. Time course experiments showed that (65)Zn efflux is shut down rapidly by DTPA in transformed cells, whereas the chelator causes greater efflux from primary hepatocytes over the first 6 h. Experiments with (14)C-labeled DTPA confirmed that this chelator does not cross cell membranes, showing that it operates entirely within the medium. Expression of ZnT-1, the efflux transporter, was not affected by DTPA in H4IIE cells. Thus, zinc deprivation enhanced zinc retention in established cell lines but increased efflux from primary cells, perhaps reflecting differing requirements for this mineral.