Trust your gut: Establishing confidence in gastrointestinal models - An overview of the state of the science and contexts of use.

The webinar series and workshop titled Trust Your Gut: Establishing Confidence in Gastrointestinal Models - An Overview of the State of the Science and Contexts of Use was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)-related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and processes occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting.

Non-animal methods to assess whether chemicals may be toxic to the human digestive tract promise to complement or improve on animal-based methods. These approaches, which are based on human or animal cells and/or computer models, are faced with their own technical challenges and need to be shown to predict adverse effects in humans. Regulators are tasked with evaluating submitted data to best protect human health and the environment. A webinar series and workshop brought together scientists from academia, industry, military, and regulatory authorities from different countries to discuss how non-animal methods can be integrated into the risk assessment of drugs, food additives, dietary supplements, pesticides, and industrial chemicals for gastrointestinal toxicity.

Safety assessment for Tris(2,4-di-tert-butylphenyl) phosphite (Irgafos 168) used as an antioxidant and stabilizer in food contact applications.

During and after fabrication of polymeric food contact articles (FCA), polymers undergo oxidation by thermal decomposition processes initiated by oxygen, heat, light, shear, and catalyst residues. To reduce degradation of the polymer, a commonly used secondary antioxidant (AO), Irgafos 168 (I-168), may be included. Use of I-168 in polymeric FCAs presents a potential concern for neurotoxicity due to its phosphate-containing degradation species, I-168ate. As a result, we evaluated dietary exposure and oral toxicity data for I-168 and its degradants when used as an AO in FCAs. Our exposure assessment included extensive review of the U.S. food-contact regulatory history of I-168 resulting in a combined cumulative estimated daily intake (CEDI) of 0.09 mg/kg bw/day for I-168 and I-168ate when used as an AO in FCAs. Our comprehensive literature review of toxicological data and supporting structure activity relationship (SAR) analysis of I-168 reactivity against acetylcholinesterase diminished concern for potential neurotoxic effects of I-168 and its degradants. An acceptable daily intake (ADI) value of 1 mg/kg bw/day for I-168 was derived from a two-year rodent combined chronic toxicity/carcinogenicity study, which is higher than the CEDI and supports the safety of current authorized food contact use levels of I-168.

Comparative analysis of the toxicological databases for 6:2 fluorotelomer alcohol (6:2 FTOH) and perfluorohexanoic acid (PFHxA).

6:2 Fluorotelomer alcohol (6:2 FTOH) is a short-chain polyfluoroalkyl substance (PFAS) in polymeric PFAS used in fast food packaging and stain- and water-resistant textiles and may be degradation products of some components of aqueous film-forming foams (AFFF). The general population is exposed to 6:2 FTOH by inhalation of evaporates from treated surfaces or ambient concentrations in air, ingestion of indoor dust, or ingestion of food packaged in materials containing PFAS. Although exposure to 6:2 FTOH is pervasive, little is known concerning human health effects of this compound. Some published risk assessments have assumed that perfluorohexanoic acid (PFHxA), a metabolite of 6:2 FTOH, adequately models the human health effects of 6:2 FTOH. Recently identified studies conducted with 6:2 FTOH and its metabolite, 5:3 acid, have provided information that enables comparison of the toxicological profiles of PFHxA and 6:2 FTOH. This article summarizes a comparative analysis of the toxicological effects of PFHxA and 6:2 FTOH in rodents to determine whether data for PFHxA adequately models potential hazards of 6:2 FTOH exposure. Our analysis demonstrates that 6:2 FTOH is significantly more toxic than PFHxA. Use of toxicological studies conducted with PFHxA to assess 6:2 FTOH exposure may significantly underestimate human health risk.

A review of dietary exposure and toxicological information on epoxidised soybean oil (ESBO) in food-contact applications.

Plasticisers have a long history of use in the industrial manufacture and processing of polymers for the purpose of increasing the flexibility and strength of the material. Approximately, 80-90% of the plasticiser market is devoted to the production of PVC, a highly versatile thermoplastic used to produce both rigid and flexible articles. Many types of plasticisers, including ortho-phthalate esters (OPE), can be added to PVC to impart flexibility. Recently, alternatives to OPEs, such as epoxy esters and aliphatic adipates, are becoming more prevalent for use in PVC-based food-contact articles. Epoxidised soybean oil (ESBO) is used as a plasticiser in flexible PVC for many food-contact articles, including food packaging and food processing equipment, from which it can potentially migrate into food and become a component of an individual's daily diet. The purpose of this review is to provide an update on the US dietary exposure and toxicological information on ESBO used in PVC-based food-contact articles. The cumulative dietary concentration (CDC) and cumulative estimated daily intake (CEDI) for ESBO from its use as a plasticiser in PVC-based food-contact articles (i.e. gaskets for glass jar lids and film wraps) was calculated to be 2.6 mg/kg (i.e. ppm) and 0.13 mg/kg bw/d, respectively, for the general population. Some regulatory agencies have reported safety levels for ESBO, and the most conservative no observed adverse effect level (NOAEL) was identified to be 100 mg/kg bw/d (i.e. 2000 ppm) based on a two-year feeding study in rats. The current CEDI is well below these levels, supporting the safe use of ESBO in food-contact applications.

Linking polymorphic p53 response elements with gene expression in airway epithelial cells of smokers and cancer risk.

Chronic cigarette smoking exposes airway epithelial cells to thousands of carcinogens, oxidants and DNA-damaging agents, creating a field of molecular injury in the airway and altering gene expression. Studies of cytologically normal bronchial epithelial cells from smokers have identified transcription-based biomarkers that may prove useful in early diagnosis of lung cancer, including a number of p53-regulated genes. The ability of p53 to regulate transcription is critical for tumor suppression, and this suggests that single-nucleotide polymorphisms (SNPs) in functional p53 binding sites (p53 response elements, or p53REs) that affect gene expression could influence susceptibility to cancer. To connect p53RE SNP genotype with gene expression and cancer risk, we identified a set of 204 SNPs in putative p53REs, and performed cis expression quantitative trait loci (eQTL) analysis, assessing associations between SNP genotypes and mRNA levels of adjacent genes in bronchial epithelial cells obtained from 44 cigarette smokers. To further test and validate these genotype-expression associations, we searched published eQTL studies from independent populations and determined that 53% (39/74) of the bronchial epithelial eQTLs were observed in at least one of other studies. SNPs in p53REs were also evaluated for effects on p53-DNA binding using a quantitative in vitro protein-DNA binding assay. Last, based on linkage disequilibrium, we found 6 p53RE SNPs associated with gene expression were identified as cancer risk SNPs by either genome-wide association studies or candidate gene studies. We provide an approach for identifying and evaluating potentially functional SNPs that may modulate the airway gene expression response to smoking and may influence susceptibility to cancers.

Reproductive toxicity in rats with crystal nephropathy following high doses of oral melamine or cyanuric acid.

The industrial chemical melamine was used in 2007 and 2008 to raise the apparent protein content in pet feed and watered down milk, respectively. Because humans may be exposed to melamine via several different routes into the human diet as well as deliberate contamination, this study was designed to characterize the effect of high dose melamine or cyanuric acid oral exposure on the pregnant animal and developing fetus, including placental transfer. Clear rectangular crystals formed following a single triazine exposure which is a different morphology from the golden spherulites caused by combined exposure or the calculi formed when melamine combines with endogenous uric acid. Crystal nephropathy, regardless of cause, induces renal failure which in turn has reproductive sequelae. Specifically, melamine alone-treated dams had increased numbers of early and late fetal deaths compared to controls or cyanuric acid-treated dams. As melamine was found in the amniotic fluid, this study confirms transfer of melamine from mammalian mother to fetus and our study provides evidence that cyanuric acid also appears in the amniotic fluid if mothers are exposed to high doses.

Performance of urinary and gene expression biomarkers in detecting the nephrotoxic effects of melamine and cyanuric acid following diverse scenarios of co-exposure.

Although standard nephrotoxicity assessments primarily detect impaired renal function, KIM-1, clusterin, NGAL, osteopontin and TIMP-1 were recently identified biomarkers proposed to indicate earlier perturbations in renal integrity. The recent adulteration of infant and pet food with melamine (MEL) and structurally-related compounds revealed that co-ingestion of MEL and cyanuric acid (CYA) could form melamine-cyanurate crystals which obstruct renal tubules and induce acute renal failure. This study concurrently evaluated the ability of multiplexed urinary biomarker immunoassays and biomarker gene expression analysis to detect nephrotoxicity in F344 rats co-administered 60ppm each of MEL and CYA in feed or via gavage for 28days. The biomarkers were also evaluated for the ability to differentiate the effects of the compounds when co-administered using diverse dosing schedules (i.e., consecutive vs. staggered gavage) and dosing matrixes (i.e., feed vs. gavage). Our results illustrate the ability of both methods to detect and differentiate the severity of adverse effects in the staggered and consecutive gavage groups at much lower doses than previously observed in animals co-exposed to the compounds in feed. We also demonstrate that these urinary biomarkers outperform traditional diagnostic methods and represent a powerful, non-invasive indicator of chemical-induced nephrotoxicity prior to the onset of renal dysfunction.

Timing and route of exposure affects crystal formation in melamine and cyanuric exposed male and female rats: gavage vs. feeding.

Effects of the dosing matrix and timing on the onset of renal crystal formation were evaluated in male and non-pregnant female rats (Fisher 344) exposed to both melamine (MEL) and cyanuric acid (CYA) for 28 days. Rats were fed ground feed containing 60 ppm MEL and 60 ppm CYA, (5 mg/kg bw/day equivalent), or exposed via oral gavage to carboxymethylcellulose containing 5 mg/kg bw MEL followed by 5 mg/kg bw CYA either consecutively (<1 min apart) or delayed 45 min after MEL. Staggered gavage exposure to MEL/CYA caused extensive renal crystal formation as compared to when the two compounds were administered consecutively or in feed. Treatment related effects included reduced weight gain, feed consumption, and testicular weight and increased kidney weight, water consumption and urine output. Animals from the staggered MEL/CYA gavage exposure group became ill and were removed after 9 days of exposure. Approximately 1 week after the initiation of exposure microscopic urinalysis revealed MEL/CYA crystals in both groups of gavaged animals but not in the MEL/CYA feed treatment groups. Urinary crystals were smaller (10 µm) in animals consecutively gavaged. In contrast the urinary crystals were larger (20-40 µm) and frequently clumped in the animals in the staggered gavage group.

In vitro toxicity screening of chemical mixtures using HepG2/C3A cells.

Traditional toxicological methods that utilize only single pure compounds may not accurately predict risks from substances with multiple chemical constituents. A complementary approach to conventional methodologies includes in vitro systems that assess toxicity of chemical mixtures and identify components that may adversely impact biological processes. Compared to animal models, in vitro assays are inexpensive, rapid, and reduce and refine related animal testing. We utilized HepG2/C3A cells as a hepatotoxicity screening model to evaluate the cytotoxic and metabolic effects of three commercially available oil dispersants, Corexit EC9500A and EC9527A and ZI-400. The surfactant DOSS, a primary active constituent of the Corexit dispersants, was also evaluated. Biologically relevant endpoints were measured including cell viability, oxidative stress, and mitochondrial activity. Significant increases in cytotoxicity were observed with Corexit dispersants (LC(50)∼250 ppm), whereas ZI-400 was moderately cytotoxic (LC(50) >>400 ppm). Each dispersant caused an accumulation of reactive oxygen species and altered mitochondrial activity and other cellular processes. Generally, DOSS made notable contributions to the effects of EC9500A and EC9527A, however, they were observed at concentrations higher than those used in most consumer products. Overall, this system may represent a valuable complementary tool for predicting the toxicity of complex mixtures.

Human single-nucleotide polymorphisms alter p53 sequence-specific binding at gene regulatory elements.

p53 coordinates the expression of an intricate network of genes in response to stress signals. Sequence-specific DNA binding is essential for p53-mediated tumor suppression. We evaluated the impact of single-nucleotide polymorphisms (SNPs) in p53 response elements (p53RE) on DNA binding and gene expression in response to DNA damage. Using a bioinformatics approach based on incorporating p53 binding strength into a position weight matrix, we selected 32 SNPs in putative and validated p53REs. The microsphere assay for protein-DNA binding (MAPD) and allele-specific expression analysis was employed to assess the impact of SNPs on p53-DNA binding and gene expression, respectively. Comparing activated p53 binding in nuclear extracts from doxorubicin- or ionizing radiation (IR)-treated human cells, we observed little difference in binding profiles. Significant p53 binding was observed for most polymorphic REs and several displayed binding comparable to the p21 RE. SNP alleles predicted to lower p53 binding indeed reduced binding in 25 of the 32 sequences. Chromatin immunoprecipitation-sequencing in lymphoblastoid cells confirmed p53 binding to seven polymorphic p53 REs in response to doxorubicin. In addition, five polymorphisms were associated with altered gene expression following doxorubicin treatment. Our findings demonstrate an effective strategy to identify and evaluate SNPs that may alter p53-mediated stress responses.

Formation of stress-specific p53 binding patterns is influenced by chromatin but not by modulation of p53 binding affinity to response elements.

The p53 protein is crucial for adapting programs of gene expression in response to stress. Recently, we revealed that this occurs partly through the formation of stress-specific p53 binding patterns. However, the mechanisms that generate these binding patterns remain largely unknown. It is not established whether the selective binding of p53 is achieved through modulation of its binding affinity to certain response elements (REs) or via a chromatin-dependent mechanism. To shed light on this issue, we used a microsphere assay for protein-DNA binding to measure p53 binding patterns on naked DNA. In parallel, we measured p53 binding patterns within chromatin using chromatin immunoprecipitation and DNase I coupled to ligation-mediated polymerase chain reaction footprinting. Through this experimental approach, we revealed that UVB and Nutlin-3 doses, which lead to different cellular outcomes, induce similar p53 binding patterns on naked DNA. Conversely, the same treatments lead to stress-specific p53 binding patterns on chromatin. We show further that altering chromatin remodeling using an histone acetyltransferase inhibitor reduces p53 binding to REs. Altogether, our results reveal that the formation of p53 binding patterns is not due to the modulation of sequence-specific p53 binding affinity. Rather, we propose that chromatin and chromatin remodeling are required in this process.

Cleavage of plasmid DNA by eukaryotic topoisomerase II.

Topoisomerase II is an essential enzyme that is required for a number of critical nuclear processes. All of the catalytic functions of topoisomerase II require the enzyme to generate a transient double-stranded break in the backbone of the double helix. To maintain genomic integrity during the cleavage event, topoisomerase II forms covalent bonds between active site tyrosyl residues and the newly generated 5'-DNA termini. In addition to the critical cellular functions of the type II enzyme, several important anticancer drugs kill cells by increasing levels of covalent topoisomerase II-DNA cleavage complexes. Due to the physiological importance of topoisomerase II and its role in cancer chemotherapy, several methods have been developed to monitor the in vitro DNA cleavage activity of the type II enzyme. The plasmid-based system described in this chapter quantifies enzyme-mediated double-stranded DNA cleavage by monitoring the conversion of covalently closed supercoiled DNA to linear molecules. The assay is simple, straightforward, and does not require the use of radiolabeled substrates.

Probing the functional impact of sequence variation on p53-DNA interactions using a novel microsphere assay for protein-DNA binding with human cell extracts.

The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs). Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation -- including polymorphisms -- and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt) variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks.

The efficacy of topoisomerase II-targeted anticancer agents reflects the persistence of drug-induced cleavage complexes in cells.

Genistein, a widely consumed bioflavonoid with chemopreventative properties in adults, and etoposide, a commonly prescribed anticancer drug, are well-characterized topoisomerase II poisons. Although both compounds display similar potencies against human topoisomerase IIalpha and IIbeta in vitro and induce comparable levels of DNA cleavage complexes in cultured human cells, their cytotoxic and genotoxic effects differ significantly. As determined by assays that monitored cell viability or the phosphorylation of histone H2AX, etoposide was much more toxic in CEM cells than genistein. Further studies that characterized the simultaneous treatment of cells with genistein and etoposide indicate that the differential actions of the two compounds are not related to the effects of genistein on cellular processes outside of its activity against topoisomerase II. Rather, they appear to result from a longer persistence of cleavage complexes induced by etoposide as compared to genistein. Parallel in vitro studies with purified type II enzymes led to similar conclusions regarding cleavage complex persistence. Isoform-specific differences were observed in vitro and in cells treated with etoposide. To this point, the t 1/2 of etoposide-induced DNA cleavage complexes formed with topoisomerase IIalpha in CEM cells was approximately 5 times longer than those formed with topoisomerase IIbeta. The cytotoxicity of etoposide following four treatment-recovery cycles was similar to that induced by continuous exposure to the drug over an equivalent time period. Taken together, these findings suggest that it may be possible to preferentially target topoisomerase IIalpha with etoposide by employing a schedule that utilizes pulsed drug treatment-recovery cycles.

Dietary polyphenols as topoisomerase II poisons: B ring and C ring substituents determine the mechanism of enzyme-mediated DNA cleavage enhancement.

Dietary polyphenols are a diverse and complex group of compounds that are linked to human health. Many of their effects have been attributed to the ability to poison (i.e., enhance DNA cleavage by) topoisomerase II. Polyphenols act against the enzyme by at least two different mechanisms. Some compounds are traditional, redox-independent topoisomerase II poisons, interacting with the enzyme in a noncovalent manner. Conversely, others enhance DNA cleavage in a redox-dependent manner that requires covalent adduction to topoisomerase II. Unfortunately, the structural elements that dictate the mechanism by which polyphenols poison topoisomerase II have not been identified. To resolve this issue, the activities of two classes of polyphenols against human topoisomerase IIalpha were examined. The first class was a catechin series, including (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC). The second was a flavonol series, including myricetin, quercetin, and kaempferol. Compounds were categorized into four distinct groups: EGCG and EGC were redox-dependent topoisomerase II poisons, kaempferol and quercetin were traditional poisons, myricetin utilized both mechanisms, and ECG and EC displayed no significant activity. On the basis of these findings, a set of rules is proposed that predicts the mechanism of bioflavonoid action against topoisomerase II. The first rule centers on the B ring. While the C4'-OH is critical for the compound to act as a traditional poison, the addition of -OH groups at C3' and C5' increases the redox activity of the B ring and allows the compound to act as a redox-dependent poison. The second rule centers on the C ring. The structure of the C ring in the flavonols is aromatic and planar and includes a C4-keto group that allows the formation of a proposed pseudo ring with the C5-OH. Disruption of these elements abrogates enzyme binding and precludes the ability to function as a traditional topoisomerase II poison.

(-)-Epigallocatechin gallate, a major constituent of green tea, poisons human type II topoisomerases.

(-)-Epigallocatechin gallate (EGCG) is the most abundant and biologically active polyphenol in green tea, and many of the therapeutic benefits of the beverage have been attributed to this compound. High concentrations of EGCG are cytotoxic and trigger genotoxic events in mammalian cells. Although this catechin affects a number of cellular systems, the genotoxic effects of several bioflavonoid-based dietary polyphenols are believed to be mediated, at least in part, by their actions on topoisomerase II. Therefore, the effects of green tea extract and EGCG on DNA cleavage mediated by human topoisomerase IIalpha and beta were characterized. The extract and EGCG increased levels of DNA strand breaks generated by both enzyme isoforms. However, EGCG acted by a mechanism that was distinctly different from those of genistein, a dietary polyphenol, and etoposide, a widely prescribed anticancer drug. In contrast to these agents, EGCG exhibited all of the characteristics of a redox-dependent topoisomerase II poison that acts by covalently adducting to the enzyme. First, EGCG stimulated DNA scission mediated by both isoforms primarily at sites that were cleaved in the absence of compounds. Second, exposure of EGCG to the reducing agent dithiothreitol (DTT) prior to its addition to DNA cleavage assays abrogated the effects of the catechin on DNA scission. Third, once EGCG stimulated topoisomerase II-mediated DNA cleavage, exposure to DTT did not effect levels of DNA strand breaks. Finally, EGCG inhibited the DNA cleavage activities of topoisomerase IIalpha and beta when incubated with either enzyme prior to the addition of DNA. Taken together, these results provide strong evidence that EGCG is a redox-dependent topoisomerase II poison and utilizes a mechanism similar to that of 1,4-benzoquinone.

Bioflavonoids as poisons of human topoisomerase II alpha and II beta.

Bioflavonoids are human dietary components that have been linked to the prevention of cancer in adults and the generation of specific types of leukemia in infants. While these compounds have a broad range of cellular activities, many of their genotoxic effects have been attributed to their actions as topoisomerase II poisons. However, the activities of bioflavonoids against the individual isoforms of human topoisomerase II have not been analyzed. Therefore, we characterized the activity and mechanism of action of three major classes of bioflavonoids, flavones, flavonols, and isoflavones, against human topoisomerase IIalpha and IIbeta. Genistein was the most active bioflavonoid tested and stimulated enzyme-mediated DNA cleavage approximately 10-fold. Generally, compounds were more active against topoisomerase IIbeta. DNA cleavage with both enzyme isoforms required a 5-OH and a 4'-OH and was enhanced by the presence of additional hydroxyl groups on the pendant ring. Competition DNA cleavage and topoisomerase II binding studies indicate that the 5-OH group plays an important role in mediating genistein binding, while the 4'-OH moiety contributes primarily to bioflavonoid function. Bioflavonoids do not require redox cycling for activity and function primarily by inhibiting enzyme-mediated DNA ligation. Mutagenesis studies suggest that the TOPRIM region of topoisomerase II plays a role in genistein binding. Finally, flavones, flavonols, and isoflavones with activity against purified topoisomerase IIalpha and IIbeta enhanced DNA cleavage by both isoforms in human CEM leukemia cells. These data support the hypothesis that bioflavonoids function as topoisomerase II poisons in humans and provide a framework for further analysis of these important dietary components.