Trends in ingredients added to infant formula: FDA's experiences in the GRAS notification program.

While human milk is considered the optimal source of nutrition for infants for the first six and twelve months of age, with continued benefit of breastfeeding with complementary foods, a safe alternative, nutritionally adequate to support infant growth and development, is necessary. In the United States, the Food and Drug Administration (FDA) establishes the requirements necessary to demonstrate the safety of infant formula within the framework of the Federal Food, Drug, and Cosmetic Act. FDA's Center for Food Safety and Applied Nutrition/Office of Food Additive Safety evaluates the safety and lawfulness of individual ingredients used in infant formula, whereas the Office of Nutrition and Food Labeling oversees the safety of infant formula. Most infant formula ingredients are either from sources with history of safe consumption by infants or are like components in human milk. Information demonstrating the regulatory status of all ingredients is required in submissions for new infant formulas, and ingredient manufacturers often use the Generally Recognized as Safe (GRAS) Notification program to establish ingredient regulatory status. We provide an overview of ingredients used in infant formula evaluated through the GRAS Notification program to highlight trends and discuss the data and information used to reach these GRAS conclusions.

Dietary exposures for the safety assessment of seven emulsifiers commonly added to foods in the United States and implications for safety.

Dietary exposure assessment using food-consumption data and ingredient-use level is essential for assessing the safety of food ingredients. Dietary exposure estimates are compared with safe intake levels, such as the acceptable daily intake (ADI). The ADI is estimated by applying a safety factor to an experimentally determined no-observed-adverse-effect level of a test substance. Two food ingredients classified as emulsifiers, carboxymethylcellulose (CMC) and polysorbate 80 (P80), received attention recently due to their putative adverse effects on gut microbiota. Because no published dietary exposure estimates for commonly used emulsifiers exist for the US population, the current investigation focused on the estimation of dietary exposure to seven emulsifiers: CMC, P80, lecithin, mono- and diglycerides (MDGs), stearoyl lactylates, sucrose esters, and polyglycerol polyricinoleate. Using maximum-use levels obtained from publicly available sources, dietary exposures to these emulsifiers were estimated for the US population (aged 2 years and older) for two time periods (1999-2002 and 2003-10) using 1- and 2-day food-consumption data from the National Health and Nutrition Examination Survey, and 10-14-day food-consumption data from NPD Group, Inc.'s National Eating Trends - Nutrient Intake Database. Our analyses indicated that among the emulsifiers assessed, lecithin and MDGs have the highest mean exposures at about 60 and about 80 mg kg-1 bw day-1, respectively, whereas the exposure to CMC is half to one-third that of lecithin or MDGs; and the exposure to P80 is approximately half that of CMC. The review of available safety information such as ADIs established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), in light of our updated dietary exposure estimates for these seven emulsifiers, did not raise safety concerns at the current specified levels of use. Additionally, by examining two time periods (1999-2002, 2003-10), it was concluded that there is no evidence that exposure levels to emulsifiers have substantially increased.

Geminin is Essential to Prevent DNA Re-Replication-Dependent Apoptosis in Pluripotent Cells, but not in Differentiated Cells.

Geminin is a dual-function protein unique to multicellular animals with roles in modulating gene expression and preventing DNA re-replication. Here, we show that geminin is essential at the beginning of mammalian development to prevent DNA re-replication in pluripotent cells, exemplified by embryonic stem cells, as they undergo self-renewal and differentiation. Embryonic stem cells, embryonic fibroblasts, and immortalized fibroblasts were characterized before and after geminin was depleted either by gene ablation or siRNA. Depletion of geminin under conditions that promote either self-renewal or differentiation rapidly induced DNA re-replication, followed by DNA damage, then a DNA damage response, and finally apoptosis. Once differentiation had occurred, geminin was no longer essential for viability, although it continued to contribute to preventing DNA re-replication induced DNA damage. No relationship was detected between expression of geminin and genes associated with either pluripotency or differentiation. Thus, the primary role of geminin at the beginning of mammalian development is to prevent DNA re-replication-dependent apoptosis, a role previously believed essential only in cancer cells. These results suggest that regulation of gene expression by geminin occurs only after pluripotent cells differentiate into cells in which geminin is not essential for viability.

The dual roles of geminin during trophoblast proliferation and differentiation.

Geminin is a protein involved in both DNA replication and cell fate acquisition. Although it is essential for mammalian preimplantation development, its role remains unclear. In one study, ablation of the geminin gene (Gmnn) in mouse preimplantation embryos resulted in apoptosis, suggesting that geminin prevents DNA re-replication, whereas in another study it resulted in differentiation of blastomeres into trophoblast giant cells (TGCs), suggesting that geminin regulates trophoblast specification and differentiation. Other studies concluded that trophoblast differentiation into TGCs is regulated by fibroblast growth factor-4 (FGF4), and that geminin is required to maintain endocycles. Here we show that ablation of Gmnn in trophoblast stem cells (TSCs) proliferating in the presence of FGF4 closely mimics the events triggered by FGF4 deprivation: arrest of cell proliferation, formation of giant cells, excessive DNA replication in the absence of DNA damage and apoptosis, and changes in gene expression that include loss of Chk1 with up-regulation of p57 and p21. Moreover, FGF4 deprivation of TSCs reduces geminin to a basal level that is required for maintaining endocycles in TGCs. Thus, geminin acts both like a component of the FGF4 signal transduction pathway that governs trophoblast proliferation and differentiation, and geminin is required to maintain endocycles.

TEAD4 establishes the energy homeostasis essential for blastocoel formation.

It has been suggested that during mouse preimplantation development, the zygotically expressed transcription factor TEAD4 is essential for specification of the trophectoderm lineage required for producing a blastocyst. Here we show that blastocysts can form without TEAD4 but that TEAD4 is required to prevent oxidative stress when blastocoel formation is accompanied by increased oxidative phosphorylation that leads to the production of reactive oxygen species (ROS). Both two-cell and eight-cell Tead4(-/-) embryos developed into blastocysts when cultured under conditions that alleviate oxidative stress, and Tead4(-/-) blastocysts that formed under these conditions expressed trophectoderm-associated genes. Therefore, TEAD4 is not required for specification of the trophectoderm lineage. Once the trophectoderm was specified, Tead4 was not essential for either proliferation or differentiation of trophoblast cells in culture. However, ablation of Tead4 in trophoblast cells resulted in reduced mitochondrial membrane potential. Moreover, Tead4 suppressed ROS in embryos and embryonic fibroblasts. Finally, ectopically expressed TEAD4 protein could localize to the mitochondria as well as to the nucleus, a property not shared by other members of the TEAD family. These results reveal that TEAD4 plays a crucial role in maintaining energy homeostasis during preimplantation development.

Organogenesis relies on SoxC transcription factors for the survival of neural and mesenchymal progenitors.

During organogenesis, neural and mesenchymal progenitor cells give rise to many cell lineages, but their molecular requirements for self-renewal and lineage decisions are incompletely understood. In this study, we show that their survival critically relies on the redundantly acting SoxC transcription factors Sox4, Sox11 and Sox12. The more SoxC alleles that are deleted in mouse embryos, the more severe and widespread organ hypoplasia is. SoxC triple-null embryos die at midgestation unturned and tiny, with normal patterning and lineage specification, but with massively dying neural and mesenchymal progenitor cells. Specific inactivation of SoxC genes in neural and mesenchymal cells leads to selective apoptosis of these cells, suggesting SoxC cell-autonomous roles. Tead2 functionally interacts with SoxC genes in embryonic development, and is a direct target of SoxC proteins. SoxC genes therefore ensure neural and mesenchymal progenitor cell survival, and function in part by activating this transcriptional mediator of the Hippo signalling pathway.

The acrosomal protein Dickkopf-like 1 (DKKL1) facilitates sperm penetration of the zona pellucida.

OBJECTIVE: To determine the role of Dkkl1 in mouse development, viability, and fertility. DESIGN: Prospective experimental study. SETTING: Government research institution. ANIMAL(S): Mice of C57BL/6, B6D2F1/J, and 129X1/SvJ strains, as well as transgenic mice of mixed C57BL/6 and 129X1/SvJ strains were used for the studies. INTERVENTION(S): Expression of the Dkkl1 gene was characterized during early mouse development, and the effects of Dkkl1 ablation on reproduction and fertility were characterized in vitro and in vivo. MAIN OUTCOME MEASURE(S): Dkkl1 RNA expression was determined by Northern blotting hybridization as well as quantitative reverse transcriptase-polymerase chain reaction assays. In vitro fertilization assays were used to assess fertility of sperm from male mice lacking functional Dkkl1. RESULT(S): Dkkl1 is a gene unique to mammals that is expressed primarily in developing spermatocytes and its product localized in the acrosome of mature sperm. Here we show that Dkkl1 also is expressed in the trophectoderm/placental lineage. Surprisingly, embryos lacking DKKL1 protein developed into viable, fertile adults. Nevertheless, the ability of sperm that lacked DKKL1 protein to fertilize wild-type eggs was severely compromised in vitro. Because this defect could be overcome either by removal of the zona pellucida or by the presence of wild-type sperm, Dkkl1, either directly or indirectly, facilitates the ability of sperm to penetrate the zona pellucida. Penetration of the zona pellucida by Dkkl1(-) sperm was delayed in vivo as well as in vitro, but the delay in vivo was compensated by other factors during preimplantation development. Accordingly, Dkkl1-/- males offer an in vitro fertilization model for identifying factors that may contribute to infertility. CONCLUSION(S): DKKL1 is a mammalian-specific, acrosomal protein that strongly affects in vitro fertilization, although the effect is attenuated in vivo.

The acrosomal protein Dickkopf-like 1 (DKKL1) is not essential for fertility.

OBJECTIVE: To determine the role of Dkkl1 on mouse development, viability, and fertility. DESIGN: Prospective experimental study. SETTING: Government research institution. ANIMAL(S): Mice of C57BL/6 and 129X1/SvJ strains, as well as transgenic mice of mixed C57BL/6 and 129X1/SvJ strains were used for the studies. INTERVENTION(S): Mice were constructed that lacked a functional Dkkl1 gene. MAIN OUTCOME MEASURE(S): Deletion of the gene was confirmed by DNA, RNA, and protein analyses; in vivo fertility was examined by continuous mating scheme. RESULT(S): Previous studies have shown that Dkkl1, a gene unique to mammals, is expressed predominantly, if not exclusively, in developing spermatocytes, and the DKKL1 protein accumulates in the acrosome of mature sperm. Subsequent studies (reported in the accompanying article) demonstrate that Dkkl1 also is expressed in the trophectoderm/placental lineage. Taken together, these results strongly suggested that DKKL1 protein is required for terminal differentiation either of trophoblast giant cells or of sperm, both of which are directly involved in fertility. To challenge this hypothesis, conditional targeted mutagenesis was used to ablate the Dkkl1 gene in mice. Surprisingly, Dkkl1 nullizygous embryos developed into viable, fertile adults, despite the fact that they failed to produce any portion of the DKKL1 protein. CONCLUSION(S): DKKL1 is a mammalian-specific acrosomal protein that is not essential either for development or fertility.

Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development.

Specification of cell lineages in mammals begins shortly after fertilization with formation of a blastocyst consisting of trophectoderm, which contributes exclusively to the placenta, and inner cell mass (ICM), from which the embryo develops. Here we report that ablation of the mouse Tead4 gene results in a preimplantation lethal phenotype, and TEAD4 is one of two highly homologous TEAD transcription factors that are expressed during zygotic gene activation in mouse 2-cell embryos. Tead4(-/-) embryos do not express trophectoderm-specific genes, such as Cdx2, but do express ICM-specific genes, such as Oct4 (also known as Pou5f1). Consequently, Tead4(-/-) morulae do not produce trophoblast stem cells, trophectoderm or blastocoel cavities, and therefore do not implant into the uterine endometrium. However, Tead4(-/-) embryos can produce embryonic stem cells, a derivative of ICM, and if the Tead4 allele is not disrupted until after implantation, then Tead4(-/-) embryos complete development. Thus, Tead4 is the earliest gene shown to be uniquely required for specification of the trophectoderm lineage.

Transcription factor TEAD2 is involved in neural tube closure.

TEAD2, one of the first transcription factors expressed at the beginning of mammalian development, appears to be required during neural development. For example, Tead2 expression is greatest in the dorsal neural crest where it appears to regulate expression of Pax3, a gene essential for brain development. Consistent with this hypothesis, we found that inactivation of the Tead2 gene in mice significantly increased the risk of exencephaly (a defect in neural tube closure). However, none of the embryos exhibited spina bifida, the major phenotype of Pax3 nullizygous embryos, and expression of Pax3 in E11.5 Tead2 nullizygous embryos was normal. Thus, Tead2 plays a role in neural tube closure that is independent of its putative role in Pax3 regulation. In addition, the risk of exencephaly was greatest with Tead2 nullizygous females, and could be suppressed either by folic acid or pifithrin-alpha. These results reveal a maternal genetic contribution to neural tube closure, and suggest that Tead2-deficient mice provide a model for anencephaly, a common human birth defect that can be prevented by folic acid.

DkkL1 (Soggy), a Dickkopf family member, localizes to the acrosome during mammalian spermatogenesis.

Dickkopf-like 1 (DkkL1) is related to the Dickkopf gene family, a group of proteins that are characterized as secreted antagonists of Wingless (Wnt) signal transduction proteins. DkkL1 mRNA is found in preimplantation mouse embryos and in developing neural tissue, but in adults it is found primarily in the testes. In an effort to elucidate its function, the distribution of DkkL1 protein in mouse testis and mature sperm was analyzed by immuno-histochemistry and immuno-blotting techniques. DkkL1 first appeared in the developing spermatocytes in seminiferous tubules as early as Stage XII, coincident with the appearance of DkkL1 mRNA. Surprisingly, however, DkkL1 localized to the developing acrosome in spermatocytes and spermatids and to the acrosome in mature sperm. Furthermore, DkkL1 was N-glycosylated in the testis, but it did not appear to be excreted, and the DkkL1 in mature sperm was no longer N-glycosylated, suggesting that additional post-translational modifications occurred during the final stages of spermatogenesis. These results identify a member of the Dickkopf family as a novel acrosomal protein that may be involved in acrosome assembly or function, a unique role for a secreted signaling molecule.

DNA methylation may restrict but does not determine differential gene expression at the Sgy/Tead2 locus during mouse development.

Soggy (Sgy) and Tead2, two closely linked genes with CpG islands, were coordinately expressed in mouse preimplantation embryos and embryonic stem (ES) cells but were differentially expressed in differentiated cells. Analysis of established cell lines revealed that Sgy gene expression could be fully repressed by methylation of the Sgy promoter and that DNA methylation acted synergistically with chromatin deacetylation. Differential gene expression correlated with differential DNA methylation, resulting in sharp transitions from methylated to unmethylated DNA at the open promoter in both normal cells and tissues, as well as in established cell lines. However, neither promoter was methylated in normal cells and tissues even when its transcripts were undetectable. Moreover, the Sgy promoter remained unmethylated as Sgy expression was repressed during ES cell differentiation. Therefore, DNA methylation was not the primary determinant of Sgy/Tead2 expression. Nevertheless, Sgy expression was consistently restricted to basal levels whenever downstream regulatory sequences were methylated, suggesting that DNA methylation restricts but does not regulate differential gene expression during mouse development.