Challenges of including human exposure to chemicals in food packaging as a new exposure pathway in life cycle impact assessment

Purpose

Limiting exposure to potentially toxic chemicals in food packaging can lead to environmental impact trade-offs. No available tool, however, considers trade-offs between environmental impacts of packaging systems and exposure to potentially toxic chemicals in food packaging. This study therefore explores the research needs for extending life cycle impact assessment (LCIA) to include exposure to chemicals in food packaging.

Methods

The LCIA framework for human toxicity was extended for the first time to include consumer exposure to chemicals in food packaging through the product intake fraction (PiF) metric. The related exposure pathway was added to LCIA without other modifications to the existing toxicity characterization framework used by USEtox®, i.e., effect factor derivation. The developed method was applied to a high impact polystyrene (HIPS) container case study with the functional unit of providing 1 kg of yogurt in single servings. Various exposure scenarios were considered, including an evidence-based scenario using concentration data and a migration model. Human toxicity impact scores in comparative toxic units (CTU_h) for the use stage were evaluated and then compared to human toxicity impact scores from a conventional LCIA methodology.

Results and discussion

Data allowed toxicity characterization of use stage exposure to only seven chemicals in HIPS out of fourty-four identified. Data required were the initial concentration of chemicals in food packaging, chemical mass transfer from packaging into food, and relevant toxicity information. Toxicity characterization demonstrated that the combined CTU_h for HIPS material acquisition, manufacturing, and disposal stages exceeded the toxicity scores related to consumer exposure to previously estimated concentrations of the seven characterizable chemicals in HIPS, by about two orders of magnitude. The CTU_h associated with consumer exposure became relevant when migration was above 0.1% of the European regulatory levels. Results emphasize missing data for chemical concentrations in food contact materials and a need to expand the current USEtox method for effect factor derivation (e.g., to consider endocrine disruption, mixture toxicity, background exposure, and thresholds when relevant).

Conclusions

An LCIA method was developed to include consumer exposure to chemicals in food packaging. Further study is required to assess realistic scenarios to inform decisions and policies, such as circular economy, which can lead to trade-offs between environmental impacts and potentially toxic chemicals in packaging. To apply the developed method, data regarding occurrence, concentration, and toxicity of chemicals in food packaging are needed. Revisiting the derivation of effect factors in future work could improve the interpretation of human toxicity impact scores.

     

Spatial gene expression in the T-stage mouse metanephros

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.     

Tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity

Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We previously identified several fully human neutralizing anti-VEGF receptor 2 (or kinase inserting domain-containing receptor (KDR)) antibodies from a large antibody phage display library. These antibodies bind specifically to KDR, block VEGF/KDR interaction, and inhibit VEGF-induced proliferation of human endothelial cells and migration of KDR+ leukemia cells. Three of these antibodies, interestingly, share an identical heavy chain variable (VH) sequence. In this report, we constructed a new library comprising the single VH paired with the variable light chain (VL) repertoire obtained from the original na?ve human library. Initial in vitro selection revealed that the single VH could pair with a number of different VL while retaining its specificity for KDR. However, a consensus VH/VL pair, clone 1121, was identified after three or four rounds of selection by tailoring the stringency of the panning conditions. Clone 1121 showed a >30-fold higher binding affinity to KDR (Kd, 100 pm) because of improvement on both association and dissociation constants and blocked VEGF/KDR interaction with an IC50 of approximately 1 nm, compared with that of 3-4 nm for the parent Fab fragments. Further, clone 1121 was more potent in inhibiting VEGF-stimulated KDR phosphorylation in endothelial cells. A binding epitope mapping study on clone 1121 and one of the parent clones, 2C6, demonstrated that both antibodies interacted with the third immunoglobulin domain within the extracellular region of KDR. Several peptide phage display libraries were utilized to further examine the fine binding specificities of the two antibodies. All of the 2C6-binding peptides are cysteine-constrained, whereas clone 1121 binds to both cysteine-constrained and linear peptides. It is noteworthy that most of the 2C6-binding peptides also cross-react with clone 1121, but none of the clone 1121-specific peptides binds to 2C6, indicating that clone 1121 retained part of the original binding epitope(s) of 2C6 while gaining new binding specificity. Taken together, our observation suggests that clone 1121 may have great clinical potential in anti-angiogenesis therapy. It further underscores the efforts to identify antibodies of high affinity for enhanced biological activities.     

Selective Activation of Human Dendritic Cells by OM-85 through a NF-kB and MAPK Dependent Pathway

OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®), a product made of the water soluble fractions of 21 inactivated bacterial strain patterns responsible for respiratory tract infections, is used for the prevention of recurrent upper respiratory tract infections and acute exacerbations in chronic obstructive pulmonary disease patients. OM-85 is able to potentiate both innate and adaptive immune responses. However, the molecular mechanisms responsible for OM-85 activation are still largely unknown. Purpose of this study was to investigate the impact of OM-85 stimulation on human dendritic cell functions. We show that OM-85 selectively induced NF-kB and MAPK activation in human DC with no detectable action on the interferon regulatory factor (IRF) pathway. As a consequence, chemokines (i.e. CXCL8, CXCL6, CCL3, CCL20, CCL22) and B-cell activating cytokines (i.e. IL-6, BAFF and IL-10) were strongly upregulated. OM-85 also synergized with the action of classical pro-inflammatory stimuli used at suboptimal concentrations. Peripheral blood mononuclear cells from patients with COPD, a pathological condition often associated with altered PRR expression pattern, fully retained the capability to respond to OM-85. These results provide new insights on the molecular mechanisms of OM-85 activation of the immune response and strengthen the rational for its use in clinical settings.     

Somatostatin in the physiologic feedback control of thyrotropin secretion

The addition of an antiserum against Somatostatin (SRIF) to a culture of dispersed rat pituitary cells incubated in the presence of hypothalamic tissue enhances thyrotropin (TSH) secretion into the medium. This indicates that SRIF is normally secreted in amounts sufficient to affect TSH secretion and suggests that SRIF may be the agent responsible for the unexplained rapid termination of TSH secretion by thyrotrophs which have been activated by Thyrotropin Releasing Factor (TRF).     

A Modular BAM Complex in the Outer Membrane of the α-Proteobacterium Caulobacter crescentus

Mitochondria are organelles derived from an intracellular α-proteobacterium. The biogenesis of mitochondria relies on the assembly of β-barrel proteins into the mitochondrial outer membrane, a process inherited from the bacterial ancestor. Caulobacter crescentus is an α-proteobacterium, and the BAM (β-barrel assembly machinery) complex was purified and characterized from this model organism. Like the mitochondrial sorting and assembly machinery complex, we find the BAM complex to be modular in nature. A ∼150 kDa core BAM complex containing BamA, BamB, BamD, and BamE associates with additional modules in the outer membrane. One of these modules, Pal, is a lipoprotein that provides a means for anchorage to the peptidoglycan layer of the cell wall. We suggest the modular design of the BAM complex facilitates access to substrates from the protein translocase in the inner membrane.     

Protein secretion and outer membrane assembly in Alphaproteobacteria

The assembly of beta-barrel proteins into membranes is a fundamental process that is essential in Gram-negative bacteria, mitochondria and plastids. Our understanding of the mechanism of beta-barrel assembly is progressing from studies carried out in Escherichia coli and Neisseria meningitidis. Comparative sequence analysis suggests that while many components mediating beta-barrel protein assembly are conserved in all groups of bacteria with outer membranes, some components are notably absent. The Alphaproteobacteria in particular seem prone to gene loss and show the presence or absence of specific components mediating the assembly of beta-barrels: some components of the pathway appear to be missing from whole groups of bacteria (e.g. Skp, YfgL and NlpB), other proteins are conserved but are missing characteristic domains (e.g. SurA). This comparative analysis is also revealing important structural signatures that are vague unless multiple members from a protein family are considered as a group (e.g. tetratricopeptide repeat (TPR) motifs in YfiO, beta-propeller signatures in YfgL). Given that the process of the beta-barrel assembly is conserved, analysis of outer membrane biogenesis in Alphaproteobacteria, the bacterial group that gave rise to mitochondria, also promises insight into the assembly of beta-barrel proteins in eukaryotes.     

Inhibition of calmodulin-dependent kinase kinase blocks human cytomegalovirus-induced glycolytic activation and severely attenuates production of viral progeny

Viruses depend on the host cell to provide the energy and biomolecular subunits necessary for production of viral progeny. We have previously reported that human cytomegalovirus (HCMV) infection induces dramatic changes to central carbon metabolism, including glycolysis, the tricarboxylic acid (TCA) cycle, fatty acid biosynthesis, and nucleotide biosynthesis. Here, we explore the mechanisms involved in HCMV-mediated glycolytic activation. We find that HCMV virion binding and tegument protein delivery are insufficient for HCMV-mediated activation of glycolysis. Viral DNA replication and late-gene expression, however, are not required. To narrow down the list of cellular pathways important for HCMV-medicated activation of glycolysis, we utilized pharmaceutical inhibitors to block pathways reported to be both involved in metabolic control and activated by HCMV infection. We find that inhibition of calmodulin-dependent kinase kinase (CaMKK), but not calmodulin-dependent kinase II (CaMKII) or protein kinase A (PKA), blocks HCMV-mediated activation of glycolysis. HCMV infection was also found to target calmodulin-dependent kinase kinase 1 (CaMKK1) expression, increasing the levels of CaMKK1 mRNA and protein. Our results indicate that inhibition of CaMKK has a negligible impact on immediate-early-protein accumulation yet severely attenuates production of HCMV viral progeny, reduces expression of at least one early gene, and blocks viral DNA replication. Inhibition of CaMKK did not affect the glycolytic activation induced by another herpes virus, herpes simplex virus type 1 (HSV-1). Furthermore, inhibition of CaMKK had a much smaller impact on HSV-1 replication than on that of HCMV. These data suggest that the role of CaMKK during the viral life cycle is, in this regard, HCMV specific. Taken together, our results suggest that CaMKK is an important factor for HCMV replication and HCMV-mediated glycolytic activation.     

The role of the microenvironment in tumor immune surveillance

The evidence appears compelling that the microenvironment, and associated biological cellular and molecular factors, may contribute to the progression of a variety of tumors. The effects of the microenvironment may directly influence the plasticity of T cell lineages, which was recently discussed (O''Shea & Paul, 2010 [4]). To review the putative role of the microenvironment in modulating the commitment of tumor immune surveillance, we use the model of oral premalignant lesions.