Species differences in specific ligand-binding affinity and activation of AHR: The biological basis for calculation of relative effective potencies and toxic equivalence factors.

In 2022 the World Health Organization (WHO) published updated 'Toxic Equivalence Factors' (TEFs) for a wide variety of chlorinated dioxins, dibenzofurans and PCBs [collectively referred to as 'dioxin-like chemicals'; DLCs) that interact with the aryl hydrocarbon receptor (AHR)]. Their update used sophisticated statistical analysis of hundreds of published studies that reported estimation of 'Relative Effective Potency' (REP) values for individual DLC congeners. The weighting scheme used in their assessment of each study favored in vivo over in vitro studies and was based largely on rodent studies. In this Commentary, we highlight the large body of published studies that demonstrate large species differences in AHR-ligand activation and provide supporting evidence for our position that the WHO 2022 TEF values intended for use in human risk assessment of DLC mixtures will provide highly misleading overestimates of 'Toxic Equivalent Quotients' (TEQs), because of well-recognized striking differences in AHR ligand affinities between rodent (rat, mouse) and human. The data reviewed in our Commentary support the position that human tissue-derived estimates of REP/TEF values for individual DLC congeners, although uncertain, will provide much better, more realistic estimates of potential activation of the human AHR, when exposure to complex DLC mixtures occurs.

Dose Range-Finding Toxicity Study in Rats With Recombinant Human Lactoferrin Produced in Komagataella phaffii.

The oral toxicity of recombinant human lactoferrin (rhLF, Helaina rhLF, Effera™) produced in Komagataella phaffii was investigated in adult Sprague Dawley rats by once daily oral gavage for 14 consecutive days. The study used groups of 3-6 rats/sex/dose. The vehicle control group received sodium citrate buffer, and the test groups received daily doses of 200, 1000, and 2000 mg of rhLF in sodium citrate buffer per kg body weight. Bovine LF at 2000 mg/kg body weight per day was used as a comparative control. Clinical observations, body weight, hematology, clinical chemistry, iron parameters, immunophenotyping, and gross examination at necropsy were used as criteria for detecting the effects of treatment in all groups and to help select dose levels for future toxicology studies. Quantitative LF levels were also analyzed as an indication of bioavailability. Overall, administration of Helaina rhLF by once daily oral gavage for 14 days was well tolerated in rats at levels up to 2000 mg/kg/day, or 57 × Helaina's intended commercial use in adults, and indicating that a high dose of 2000 mg/kg/day is appropriate for future definitive toxicology studies.

US regulations to curb alleged cancer causes are ineffectual and compromised by scientific, constitutional and ethical violations.

The 1958 Delaney amendment to the Federal Food Drug and Cosmetics Act prohibited food additives causing cancer in animals by appropriate tests. Regulators responded by adopting chronic lifetime cancer tests in rodents, soon challenged as inappropriate, for they led to very inconsistent results depending on the subjective choice of animals, test design and conduct, and interpretive assumptions. Presently, decades of discussions and trials have come to conclude it is impossible to translate chronic animal data into verifiable prospects of cancer hazards and risks in humans. Such conclusion poses an existential crisis for official agencies in the US and abroad, which for some 65 years have used animal tests to justify massive regulations of alleged human cancer hazards, with aggregated costs of $trillions and without provable evidence of public health advantages. This article addresses suitable remedies for the US and potentially worldwide, by critically exploring the practices of regulatory agencies vis-á-vis essential criteria for validating scientific evidence. According to this analysis, regulations of alleged cancer hazards and risks have been and continue to be structured around arbitrary default assumptions at odds with basic scientific and legal tests of reliable evidence. Such practices raise a manifold ethical predicament for being incompatible with basic premises of the US Constitution, and with the ensuing public expectations of testable truth and transparency from government agencies. Potential remedies in the US include amendments to the US Administrative Procedures Act, preferably requiring agencies to justify regulations compliant with the Daubert opinion of the Daubert ruling of the US Supreme Court, which codifies the criteria defining reliable scientific evidence. International reverberations are bound to follow what remedial actions may be taken in the US, the origin of current world regulatory procedures to control alleged cancer causing agents.

SLAMF7 Is a Critical Negative Regulator of IFN-α-Mediated CXCL10 Production in Chronic HIV Infection.

Current advances in combined antiretroviral therapy have rendered HIV infection a chronic, manageable disease; however, the problem of persistent immune activation still remains despite treatment. The immune cell receptor SLAMF7 has been shown to be upregulated in diseases characterized by chronic immune activation. In this study, we studied the function of the SLAMF7 receptor in immune cells of HIV patients and the impacts of SLAMF7 signaling on peripheral immune activation. We observed increased frequencies of SLAMF7+ PBMCs in HIV+ individuals in a clinical phenotype-dependent manner, with discordant and long-term nonprogressor patients showing elevated SLAMF7 levels, and elite controllers showing levels comparable to healthy controls. We also noted that SLAMF7 was sensitive to IFN-⍺ stimulation, a factor elevated during HIV infection. Further studies revealed SLAMF7 to be a potent inhibitor of the monocyte-derived proinflammatory chemokine CXCL10 (IP-10) and other CXCR3 ligands, except in a subset of HIV+ patients termed SLAMF7 silent (SF7S). Studies utilizing small molecule inhibitors revealed that the mechanism of CXCL10 inhibition is independent of known SLAMF7 binding partners. Furthermore, we determined that SLAMF7 activation on monocytes is able to decrease their susceptibility to HIV-1 infection in vitro via downregulation of CCR5 and upregulation of the CCL3L1 chemokine. Finally, we discovered that neutrophils do not express SLAMF7, are CXCL10+ at baseline, are able to secrete CXCL10 in response to IFN-⍺ and LPS, and are nonresponsive to SLAMF7 signaling. These findings implicate the SLAMF7 receptor as an important regulator of IFN-⍺-driven innate immune responses during HIV infection.

Gene co-regulation and co-expression in the aryl hydrocarbon receptor-mediated transcriptional regulatory network in the mouse liver.

Four decades after its discovery, the aryl hydrocarbon receptor (AHR), a ligand-inducible transcription factor (TF) activated by the persistent environmental contaminant 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), remains an enigmatic molecule with a controversial endogenous role. Here, we have assembled a global map of the AHR gene regulatory network in female C57BL/6 mice orally gavaged with 30 µg/kg of TCDD from a combination of previously published gene expression and genome-wide TF-binding data sets. Using Kohonen self-organizing maps and subspace clustering, we show that genes co-regulated by common upstream TFs in the AHR network exhibit a pattern of co-expression. Directly bound, indirectly bound, and non-genomic AHR target genes exhibit distinct expression patterns, with the directly bound targets associated with highest median expression. Interestingly, among the directly bound AHR target genes, the expression level increases with the number of AHR-binding sites in the proximal promoter regions. Finally, we show that co-regulated genes in the AHR network activate distinct groups of downstream biological processes. Although the specific findings described here are restricted to hepatic effects under short-term TCDD exposure, this work describes a generalizable approach to the reconstruction and analysis of transcriptional regulatory cascades underlying cellular stress response, revealing network hierarchy and the nature of information flow from the initial signaling events to phenotypic outcomes. Such reconstructed networks can form the basis of a new generation of quantitative adverse outcome pathways.

Δ9-Tetrahydrocannabinol Suppresses Monocyte-Mediated Astrocyte Production of Monocyte Chemoattractant Protein 1 and Interleukin-6 in a Toll-Like Receptor 7-Stimulated Human Coculture.

Cannabis is widely used in the United States, with an estimated prevalence of 9.5%. Certain cannabinoids in Cannabis sativa, Δ9-tetrahydrocannabinol (THC) in particular, possess immune-modulating and anti-inflammatory activity. Depending on the context, the anti-inflammatory activity of cannabinoids may be beneficial (e.g., in treating inflammatory diseases) or detrimental to normal immune defense against pathogens. The potential beneficial effect of cannabinoids on chronic neuroinflammation has gained recent attention. Monocyte migration to the brain has been implicated as a key event in chronic neuroinflammation and in the etiology of central nervous system diseases including viral infection (e.g., human immunodeficiency virus-associated neurocognitive disorder). In the brain, monocytes can contribute to neuroinflammation through interactions with astrocytes, including inducing astrocyte secretion of cytokines and chemokines. In a human coculture system, monocyte-derived interleukin (IL)-1ß due to Toll-like receptor 7 (TLR7) activation has been identified to promote astrocyte production of monocyte chemoattractant protein (MCP)-1 and IL-6. THC treatment of the TLR7-stimulated coculture suppressed monocyte secretion of IL-1ß, resulting in decreased astrocyte production of MCP-1 and IL-6. Furthermore, THC displayed direct inhibition of monocytes, as TLR7-stimulated monocyte monocultures treated with THC also showed suppressed IL-1ß production. The cannabinoid receptor 2 (CB2) agonist, JWH-015, impaired monocyte IL-1ß production similar to that of THC, suggesting that THC acts, in part, through CB2. THC also suppressed key elements of the IL-1ß production pathway, including IL1B mRNA levels and caspase-1 activity. Collectively, this study demonstrates that the anti-inflammatory properties of THC suppress TLR7-induced monocyte secretion of IL-1ß through CB2, which results in decreased astrocyte secretion of MCP-1 and IL-6. SIGNIFICANCE STATEMENT: Because cannabis use is highly prevalent in the United States and has putative anti-inflammatory properties, it is important to investigate the effect of cannabinoids on immune cell function. Furthermore, cannabinoids have garnered particular interest due to their potential beneficial effects on attenuating viral-induced chronic neuroinflammation. This study utilized a primary human coculture system to demonstrate that the major psychotropic cannabinoid in cannabis, Δ9-tetrahydrocannabinol, and a cannabinoid receptor-2 selective agonist suppress specific monocyte-mediated astrocyte inflammatory responses.

Suppression of CpG-ODN-mediated IFNα and TNFα response in human plasmacytoid dendritic cells (pDC) by cannabinoid receptor 2 (CB2)-specific agonists.

Plasmacytoid dendritic cells (pDC) compose 0.2-0.5% of circulating leukocytes but play a significant role in mounting host immune responses. Elevated and chronic activation of pDC are implicated in autoimmune disease like systemic lupus erythematosus and rheumatoid arthritis. Δ9-tetrahydrocannabinol (THC) is a well characterized cannabinoid with potent anti-inflammatory activity, but acceptance of THC as a treatment for autoimmune disorders has been hindered due to psychotropic activity. The psychotropic effects of THC are mediated through cannabinoid receptor 1 (CB1) expressed in the central nervous system while the immunomodulatory effects of THC result from THC binding to CB1 and CB2 on immune cells. Synthetic CB2-selective agonists have been developed to explore immune modulation by cannabinoids in the absence of psychotropic effects. The goal of these studies was to determine if the CB2-selective agonists, JWH-015 and JWH-133, have comparable efficacy to THC in modulating IFNα and TNFα responses by primary human pDC. Treatment with JWH-133 and JWH-015 inhibited CpG-induced IFNα and TNFα responses by pDC. Further, the phosphorylation of IRF7, TBK1, NFκB, and IKKγ, key events in pDC activation, were suppressed by THC, JWH-133, and JWH-015. Likewise, the phosphorylation of AKT at the S473 and T308 residues were differentially modulated by treatment with THC and both JWH compounds. Collectively, these results demonstrate the potential for CB2 targeted therapeutics for treatment of inflammatory conditions involving aberrant pDC activity.

Aryl Hydrocarbon Receptor Activation Suppresses EBF1 and PAX5 and Impairs Human B Lymphopoiesis.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates biological responses to endogenous and environmental chemical cues. Increasing evidence shows that the AHR plays physiological roles in regulating development, homeostasis, and function of a variety of cell lineages in the immune system. However, the role of AHR in human B cell development has not been investigated. Toward this end, an in vitro feeder-free human B cell developmental model system was employed using human cord blood CD34+ hematopoietic stem/progenitor cells. Using this model, we found that AHR activation by the high-affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin significantly suppressed the generation of early B cells and pro-B cells from hematopoietic stem/progenitor cells, indicating the impairment of B cell lineage specification and commitment. Addition of an AHR antagonist reversed 2,3,7,8-tetrachlorodibenzo-p-dioxin-elicited suppression of early B and pro-B cells, suggesting a role of AHR in regulating B lymphopoiesis. Gene expression analysis revealed a significant decrease in the messenger RNA level of early B cell factor 1 (EBF1) and paired box 5, two critical transcription factors directing B cell lineage specification and commitment. Additionally, binding of the ligand-activated AHR to the putative dioxin response elements in the EBF1 promoter was demonstrated by EMSAs and chromatin immunoprecipitation analysis, suggesting transcriptional regulation of EBF1 by AHR. Taken together, this study demonstrates a role for the AHR in regulating human B cell development, and it suggests that transcriptional alterations of EBF1 by the AHR are involved in the underlying mechanism.

Conflict of Interest Mitigation Procedures May Have Little Influence on the Perceived Procedural Fairness of Risk-Related Research.

Two between-subject experiments explored perceived conflict of interest (COI)-operationalized as perceived procedural unfairness-in a hypothetical public-private research partnership to study the health risks of trans fats. Perceived fairness was measured as subjects' perceptions that health researchers would be willing to listen to a range of voices and minimize bias (i.e., COI) in the context of a research project. Experiment 1 (n = 1,263) randomly assigned research subjects to a partnership that included (1) a combination of an industry partner, a university partner, and a nongovernmental organization (NGO) partner; and (2) one of three processes aimed at mitigating the potential for COI to harm the quality of the research. The procedures included an arm's-length process meant to keep the university-based research team from being influenced by the other partners, an independent advisory board to oversee the project, and a commitment to making all data and analyses openly available. The results suggest that having an industry partner has substantial negative effects on perceived fairness and that the benefit of employing a single COI-mitigation process may be relatively small. Experiment 2 (n = 1,076) assessed a partnership of (1) a university and either an NGO or industry partner and (b) zero, one, two, or three of the three COI-mitigation procedures. Results suggest there is little value in combining COI-mitigation procedures. The study has implications for those who aim to foster confidence in scientific findings for which the underlying research may benefit from industry funding.

Interferon-α-Mediated Activation of T Cells from Healthy and HIV-Infected Individuals Is Suppressed by Δ9-Tetrahydrocannabinol.

Patients with HIV routinely use medicinal cannabinoids to treat neuropathic pain, anxiety, and human immunodeficiency virus (HIV)-associated wasting. However, Δ9-tetrahydrocannabinol (THC), the primary psychoactive cannabinoid in cannabis, suppresses T-cell function and secretion of interferons, both critically important in the antiviral immune response. Interferon-α (IFNα), a key cytokine in T-cell activation and peripheral control of HIV infection, can potentiate responsiveness to interleukin-7 (IL-7), a crucial homeostatic cytokine for peripheral T-cell maintenance. The objective of this investigation was to compare the response of T cells to stimulation by IFNα and IL-7 in T cells from healthy and HIV+ donors in the absence and presence of THC. To compare T-cell responses between healthy and HIV+ donors signaling through IFNα receptor, IFNα-induced expression of IL-7α receptor (IL-7Rα), cognate signaling through IL-7R, and on IL-7-mediated T-cell proliferation were measured by flow cytometry and real-time quantitative polymerase chain reaction. CD8+ T cells from HIV+ donors showed a diminished response to IFNα-induced phosphorylated signal transducer and activator of transcription-1 activation compared with CD8+ T cells from healthy donors, whereas CD4+ T cells from HIV+ donors and healthy donors were comparable. Treatment with IFNα promoted IL-7R expression and potentiated IL-7-induced STAT5 phosphorylation to augment IL-7-mediated proliferation by T cells from healthy and HIV+ donors. Finally, HIV+ donors exhibited reduced sensitivity to THC-mediated suppression by IFNα- and IL-7-mediated stimulation compared with healthy donors. These results further support THC as being immune suppressive while identifying putatively beneficial aspects of cannabinoid-based therapies in HIV+ patients.

Characterizing Serpinb2 as a Modulator of TCDD-Induced Suppression of the B Cell.

2,3,7,8-Tetrachlordibenzo- p-dioxin (TCDD) is an environmental pollutant that can cause various toxic effects, including chloracne, metabolic syndrome, and immune suppression. Most of the toxicity associated with TCDD is mediated through activation of the aryl hydrocarbon receptor (AHR). Recent research has suggested the presence of a wide-range of interindividual variability in TCDD-mediated suppression of the Immunoglobulin-M (IgM) response across the human population. In an attempt to identify putative modifiers of AHR-mediated immunosuppression beyond the AHR, B cells were isolated from a panel of genetically diverse mouse strain to scan for modulators that drive interstrain differences in TCDD-mediated suppression of the IgM response. Results implicated a region of mouse Chromosome 1 near a gene encoding serine peptidase inhibitor, clade B, member 2 ( Serpinb2) whose human ortholog is plasminogen activator inhibitor 2 (PAI2). Further downstream analyses indicated that Serpinb2 is dysregulated by TCDD and, furthermore, that B cells from Serpinb2 -/- mice are significantly more sensitive to TCDD-mediated suppression as compared to littermate controls. This study suggests a protective role of Serpinb2 within TCDD-mediated immunosuppression and, furthermore, a novel function of Serpinb2-related activity in the IgM response.

Obfuscating transparency?

Several recent and prominent articles in Science and Nature deliberately mischaracterized the nature of genuine scientific evidence. Those articles take issue with the United States Environmental Protection Agency's recent proposal to structure its policies and rules only from studies with transparently published raw data. The articles claim it is an effort to obfuscate with transparency, by eliminating a host of studies not offering raw data. A remarkable declaration by a Science editorial is that properly trained experts can verify the scientific evidence of studies without access to raw data, We assert the Agency's proposal must be sustained. Transparency in reporting is a fundamental ethical imperative of objective scientific research justifying massive official regulations and policies. Putative hazards bereft of independent scientific evidence will continue to stoke public anxieties, calling for precautionary regulations and policies. These should rely not on spurious science but on transparent tradeoffs between the smallest exposures compatible with utility and with social perceptions of affordable precaution.

Comparative analysis of TCDD-induced AhR-mediated gene expression in human, mouse and rat primary B cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant that activates the aryl hydrocarbon receptor (AhR) resulting in altered gene expression. In vivo, in vitro, and ex vivo studies have demonstrated that B cells are directly impaired by TCDD, and are a sensitive target as evidenced by suppression of antibody responses. The window of sensitivity to TCDD-induced suppression of IgM secretion among mouse, rat and human B cells is similar. Specifically, TCDD must be present within the initial 12h post B cell stimulation, indicating that TCDD disrupts early signaling network(s) necessary for B lymphocyte activation and differentiation. Therefore, we hypothesized that TCDD treatment across three different species (mouse, rat and human) triggers a conserved, B cell-specific mechanism that is involved in TCDD-induced immunosuppression. RNA sequencing (RNA-Seq) was used to identify B cell-specific orthologous genes that are differentially expressed in response to TCDD in primary mouse, rat and human B cells. Time course studies identified TCDD-elicited differential expression of 515 human, 2371 mouse and 712 rat orthologous genes over the 24-h period. 28 orthologs were differentially expressed in response to TCDD in all three species. Overrepresented pathways enriched in all three species included cytokine-cytokine receptor interaction, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton and pathways in cancer. Differentially expressed genes functionally associated with cell-cell signaling in humans, immune response in mice, and oxidation reduction in rats. Overall, these results suggest that despite the conservation of the AhR and its signaling mechanism, TCDD elicits species-specific gene expression changes.

TCDD administered on activated carbon eliminates bioavailability and subsequent shifts to a key murine gut commensal.

Activated carbon (AC) is an increasingly attractive remediation alternative for the sequestration of dioxins at contaminated sites globally. However, the potential for AC to reduce the bioavailability of dioxins in mammals and the residing gut microbiota has received less attention. This question was partially answered in a recent study examining 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hallmark toxic responses in mice administered with TCDD sequestered by AC or freely available in corn oil by oral gavage. Results from that study support the use of AC to significantly reduce the bioavailability of TCDD to the host. Herein, we examined the bioavailability of TCDD sequestered to AC on a key murine gut commensal and the influence of AC on the community structure of the gut microbiota. The analysis included qPCR to quantify the expression of segmented filamentous bacteria (SFB) in the mouse ileum, which has responded to TCDD-induced host toxicity in previous studies and community structure via sequencing the 16S ribosomal RNA (rRNA) gene. The expression of SFB 16S rRNA gene and functional genes significantly increased with TCDD administered with corn oil vehicle. Such a response was absent when TCDD was sequestered by AC. In addition, AC appeared to have a minimal influence on murine gut community structure and diversity, affecting only the relative abundance of Lactobacillaceae and two other groups. Results of this study further support the remedial use of AC for eliminating bioavailability of TCDD to host and subsequent influence on the gut microbiome.

SHP-1 is directly activated by the aryl hydrocarbon receptor and regulates BCL-6 in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a strong AHR agonist, causes significant suppression of human B cell activation and differentiation. The current studies describe the identification of Src homology phosphatase 1 (SHP-1) encoded by the gene PTPN6 as a putative regulator of TCDD-mediated suppression of B cell activation. Shp-1 was initially identified through a genome-wide analysis of AHR binding in mouse B cells in the presence of TCDD. The binding of AHR to the PTPN6 promoter was further confirmed using electrophoretic mobility shift assays in which, specific binding of AHR was detected at four putative DRE sites within PTPN6 promoter. Time-course measurements performed in human B cells highlighted a significant increase in SHP-1 mRNA and protein levels in the presence of TCDD. The changes in the protein levels of SHP-1 were also observed in a TCDD concentration-dependent manner. The increase in SHP-1 levels was also seen to occur due to a change in early signaling events in the presence of TCDD. We have shown that BCL-6 regulates B cell activation by repressing activation marker CD80 in the presence of TCDD. TCDD-treatment led to a significant increase in the double positive (SHP-1hi BCL-6hi) population. Interestingly, treatment of naïve human B cells with SHP-1 inhibitor decreased BCL-6 protein levels suggesting possible regulation of BCL-6 by SHP-1 for the first time. Collectively, these results suggest that SHP-1 is regulated by AHR in the presence of TCDD and may, in part through BCL-6, regulate TCDD-mediated suppression of human B cell activation.

Macrophage depletion lowers blood pressure and restores sympathetic nerve α2-adrenergic receptor function in mesenteric arteries of DOCA-salt hypertensive rats.

We tested the hypothesis that vascular macrophage infiltration and O2 (-) release impairs sympathetic nerve α2-adrenergic autoreceptor (α2AR) function in mesenteric arteries (MAs) of DOCA-salt hypertensive rats. Male rats were uninephrectomized or sham operated (sham). DOCA pellets were implanted subcutaneously in uninephrectomized rats who were provided high-salt drinking water or high-salt water with apocynin. Sham rats received tap water. Blood pressure was measured using radiotelemetry. Treatment of sham and DOCA-salt rats with liposome-encapsulated clodronate was used to deplete macrophages. After 3-5, 10-13, and 18-21 days of DOCA-salt treatment, MAs and peritoneal fluid were harvested from euthanized rats. Norepinephrine (NE) release from periarterial sympathetic nerves was measured in vitro using amperometry with microelectrodes. Macrophage infiltration into MAs as well as TNF-α and p22(phox) were measured using immunohistochemistry. Peritoneal macrophage activation was measured by flow cytometry. O2 (-) was measured using dihydroethidium staining. Hypertension developed over 28 days, and apocynin reduced blood pressure on days 18-21. O2 (-) and macrophage infiltration were greater in DOCA-salt MAs compared with sham MAs after day 10. Peritoneal macrophage activation occurred after day 10 in DOCA-salt rats. Macrophages expressing TNF-α and p22(phox) were localized near sympathetic nerves. Impaired α2AR function and increased NE release from sympathetic nerves occurred in MAs from DOCA-salt rats after day 18. Macrophage depletion reduced blood pressure and vascular O2 (-) while restoring α2AR function in DOCA-salt rats. Macrophage infiltration into the vascular adventitia contributes to increased blood pressure in DOCA-salt rats by releasing O2 (-), which disrupts α2AR function, causing enhanced NE release from sympathetic nerves.

Utilization of a novel human hepatocyte-endothelial cell coculture model to determine differential toxicities of pyrrolizidine alkaloid food contaminants.

Pyrrolizidine alkaloids (PA) are comprised of a family of hundreds of metabolites, produced by plants as a mechanism to protect against herbivory. Upon ingestion and metabolism, dehydropyrrolizidine alkaloids are formed, which are known to generate DNA adducts and subsequently double-strand DNA breaks. Within the liver, the most sensitive cell type to PA exposure is the sinusoidal endothelial cell, as evidenced by the generation of veno-occlusive disease in the human population. PAs are a common crop contaminant and have been regulated by some agencies, using the precautionary principle; each equally potent and genotoxic. Therefore, as a proof of principle we have established a human in vitro coculture model system, utilizing the metabolically active HepaRG hepatocyte and the SK-Hep-1 endothelial cell, to determine differential potencies of different PAs commonly found in crops and food products, notably cell death, targeting of endothelial cells, and genotoxicity comparing the micronucleus assay versus γH2AX assay. Our results demonstrate differential potencies of the PAs used, which encompass three esterification states (monoester, cyclic diester, and open-chain diester). The results suggest that a more nuanced approach to the regulation of PAs may be more appropriate in the regulatory decision-making process.