Genetic Variability Impacts Genotoxic and Transcriptome Responses in the Human Colon after the Consumption of Processed Red Meat Products and Those with Added Phytochemical Extracts.

The PHYTOME study investigated the effect of consuming processed meat products on outcomes related to colorectal cancer risk without testing the impact of genetic variability on these responses. This research aims to elucidate the genetic impact on apparent total N-nitroso compound (ATNC) excretion, colonic DNA adduct formation, ex vivo-induced DNA damage, and gene expression changes in colon biopsies of healthy participants. Through a systematic literature review, candidate polymorphisms were selected and then detected using TaqMan and PCR analysis. The effect of genotype on study outcomes was determined via a linear mixed model and analysis of variance. Machine learning was used to evaluate relative allele importance concerning genotoxic responses, which established a ranking of the most protective alleles and a combination of genotypes (gene scores). Participants were grouped by GSTM1 genotype and differentially expressed genes (DEGs), and overrepresented biological pathways were compared between groups. Stratifying participants by ten relevant genes revealed significant variations in outcome responses. After consumption of processed red meat, variations in NQO1 and COMT impacted responses in ATNC levels (µmol/L) (+9.56 for wildtype vs. heterozygous) and DNA adduct levels (pg/µg DNA) (+1.26 for variant vs. wildtype and +0.43 for variant vs. heterozygous), respectively. After phytochemicals were added to the meat, GSTM1 variation impacted changes in DNA adduct levels (-6.12 for deletion vs. wildtype). The gene scores correlated with these responses and DEGs were identified by GSTM1 genotype. The altered pathways specific to the GSTM1 wildtype group included 'metabolism', 'cell cycle', 'vitamin D receptor', and 'metabolism of water-soluble vitamins and co-factors'. Genotype impacted both the potential genotoxicity of processed red meat and the efficacy of protective phytochemical extracts.

Correction: Bischoff et al. The Effects of the Food Additive Titanium Dioxide (E171) on Tumor Formation and Gene Expression in the Colon of a Transgenic Mouse Model for Colorectal Cancer. Nanomaterials 2022, 12, 1256.

In the published publication [...].

Analysis of cell-specific transcriptional responses in human colon tissue using CIBERSORTx.

Diet is an important determinant of overall health, and has been linked to the risk of various cancers. To understand the mechanisms involved, transcriptomic responses from human intervention studies are very informative. However, gene expression analysis of human biopsy material only represents the average profile of a mixture of cell types that can mask more subtle, but relevant cell-specific changes. Here, we use the CIBERSORTx algorithm to generate single-cell gene expression from human multicellular colon tissue. We applied the CIBERSORTx to microarray data from the PHYTOME study, which investigated the effects of different types of meat on transcriptional and biomarker changes relevant to colorectal cancer (CRC) risk. First, we used single-cell mRNA sequencing data from healthy colon tissue to generate a novel signature matrix in CIBERSORTx, then we determined the proportions and gene expression of each separate cell type. After comparison, cell proportion analysis showed a continuous upward trend in the abundance of goblet cells and stem cells, and a continuous downward trend in transit amplifying cells after the addition of phytochemicals in red meat products. The dietary intervention influenced the expression of genes involved in the growth and division of stem cells, the metabolism and detoxification of enterocytes, the translation and glycosylation of goblet cells, and the inflammatory response of innate lymphoid cells. These results show that our approach offers novel insights into the heterogeneous gene expression responses of different cell types in colon tissue during a dietary intervention.

Impact of Processing Method and Storage Time on Phytochemical Concentrations in an Antioxidant-Rich Food Mixture.

Foods high in phytochemicals are known for their role in the prevention of chronic disease development, but after processing and storage, such food products may lose part of their functionality as these compounds are sensitive to the impact of processing temperature and the type of methods applied. Therefore, we measured the levels of vitamin C, anthocyanins, carotenoids, catechins, chlorogenic acid, and sulforaphane in a complex blend of fruits and vegetables, and when applied to a dry food product, after exposure to different processing methods. These levels were compared between pasteurized, pascalized (high-pressure processing), and untreated conditions. Furthermore, we established the effect of freezing and storage time on the stability of these compounds. The results showed that pascalization better preserved vitamin C and sulforaphane, whereas pasteurization resulted in higher concentrations of chlorogenic acid, carotenoids, and catechins. For samples which were frozen and thawed immediately after processing, pascalization was the optimal treatment for higher contents of lutein, cyanidin-3-glucoside, quercetin-3-glucoside, delphinidin-3-glucoside, peonidin-3-glucoside, and epicatechin gallate. Ultimately, the optimal processing method to preserve phytochemicals in fruit and vegetable products is as complex as the blend of compounds, and this decision-making would best be led by the prioritized nutrient aim of an antioxidant food product.

Reply to Kaminski, N.E.; Cohen, S.M. Comment on "Bischoff et al. The Effects of the Food Additive Titanium Dioxide (E171) on Tumor Formation and Gene Expression in the Colon of a Transgenic Mouse Model for Colorectal Cancer. Nanomaterials 2022, 12, 1256".

We appreciate the interest in our article describing transcriptome changes in a transgenic mouse model carrying an APC gene mutation and would like to reply to the reader [...].

The Effects of the Food Additive Titanium Dioxide (E171) on Tumor Formation and Gene Expression in the Colon of a Transgenic Mouse Model for Colorectal Cancer.

Titanium dioxide (TiO2) is present in many different food products as the food additive E171, which is currently scrutinized due to its potential adverse effects, including the stimulation of tumor formation in the gastrointestinal tract. We developed a transgenic mouse model to examine the effects of E171 on colorectal cancer (CRC), using the Cre-LoxP system to create an Apc-gene-knockout model which spontaneously develops colorectal tumors. A pilot study showed that E171 exposed mice developed colorectal adenocarcinomas, which were accompanied by enhanced hyperplasia in epithelial cells, lymphatic nodules at the base of the polyps, and increased tumor size. In the main study, tumor formation was studied following the exposure to 5 mg/kgbw/day of E171 for 9 weeks (Phase I). E171 exposure showed a statistically nonsignificant increase in the number of colorectal tumors in these transgenic mice, as well as a statistically nonsignificant increase in the average number of mice with tumors. Gene expression changes in the colon were analyzed after exposure to 1, 2, and 5 mg/kgbw/day of E171 for 2, 7, 14, and 21 days (Phase II). Whole-genome mRNA analysis revealed the modulation of genes in pathways involved in the regulation of gene expression, cell cycle, post-translational modification, nuclear receptor signaling, and circadian rhythm. The processes associated with these genes might be involved in the enhanced tumor formation and suggest that E171 may contribute to tumor formation and progression by modulation of events related to inflammation, activation of immune responses, cell cycle, and cancer signaling.

Replacement of Nitrite in Meat Products by Natural Bioactive Compounds Results in Reduced Exposure to N-Nitroso Compounds: The PHYTOME Project.

SCOPE: It has been proposed that endogenously form N-nitroso compounds (NOCs) are partly responsible for the link between red meat consumption and colorectal cancer (CRC) risk. As nitrite has been indicated as critical factor in the formation of NOCs, the impact of replacing the additive sodium nitrite (E250) by botanical extracts in the PHYTOME project is evaluated. METHOD AND RESULTS: A human dietary intervention study is conducted in which healthy subjects consume 300 g of meat for 2 weeks, in subsequent order: conventional processed red meat, white meat, and processed red meat with standard or reduced levels of nitrite and added phytochemicals. Consumption of red meat products enriched with phytochemicals leads to a significant reduction in the faecal excretion of NOCs, as compared to traditionally processed red meat products. Gene expression changes identify cell proliferation as main affects molecular mechanism. High nitrate levels in drinking water in combination with processed red meat intake further stimulates NOC formation, an effect that could be mitigated by replacement of E250 by natural plant extracts. CONCLUSION: These findings suggest that addition of natural extracts to conventionally processed red meat products may help to reduce CRC risk, which is mechanistically support by gene expression analyses.

Possible Adverse Effects of Food Additive E171 (Titanium Dioxide) Related to Particle Specific Human Toxicity, Including the Immune System.

Titanium dioxide (TiO2) is used as a food additive (E171) and can be found in sauces, icings, and chewing gums, as well as in personal care products such as toothpaste and pharmaceutical tablets. Along with the ubiquitous presence of TiO2 and recent insights into its potentially hazardous properties, there are concerns about its application in commercially available products. Especially the nano-sized particle fraction (<100 nm) of TiO2 warrants a more detailed evaluation of potential adverse health effects after ingestion. A workshop organized by the Dutch Office for Risk Assessment and Research (BuRO) identified uncertainties and knowledge gaps regarding the gastrointestinal absorption of TiO2, its distribution, the potential for accumulation, and induction of adverse health effects such as inflammation, DNA damage, and tumor promotion. This review aims to identify and evaluate recent toxicological studies on food-grade TiO2 and nano-sized TiO2 in ex-vivo, in-vitro, and in-vivo experiments along the gastrointestinal route, and to postulate an Adverse Outcome Pathway (AOP) following ingestion. Additionally, this review summarizes recommendations and outcomes of the expert meeting held by the BuRO in 2018, in order to contribute to the hazard identification and risk assessment process of ingested TiO2.

Impact of high drinking water nitrate levels on the endogenous formation of apparent N-nitroso compounds in combination with meat intake in healthy volunteers.

BACKGROUND: Nitrate is converted to nitrite in the human body and subsequently can react with amines and amides in the gastrointestinal tract to form N-nitroso compounds (NOCs), which are known to be carcinogenic in animals. Humans can be exposed to nitrate via consumption of drinking water and diet, especially green leafy vegetables and cured meat. The contribution of nitrate from drinking water in combination with meat intake has not been investigated thoroughly. Therefore, in the present pilot study, we examined the effect of nitrate from drinking water, and its interaction with the consumption of white and processed red meat, on the endogenous formation of NOCs, taking into account the intake of vitamin C, a nitrosation inhibitor. METHODS: Twenty healthy subjects were randomly assigned to two groups consuming either 3.75 g/kg body weight (maximum 300 g per day) processed red meat or unprocessed white meat per day for two weeks. Drinking water nitrate levels were kept low during the first week (< 1.5 mg/L), whereas in week 2, nitrate levels in drinking water were adjusted to the acceptable daily intake level of 3.7 mg/kg bodyweight. At baseline, after 1 and 2 weeks, faeces and 24 h urine samples were collected for analyses of nitrate, apparent total N-nitroso compounds (ATNC), compliance markers, and genotoxic potential in human colonic Caco-2 cells. RESULTS: Urinary nitrate excretion was significantly increased during the high drinking water nitrate period for both meat types. Furthermore, levels of compliance markers for meat intake were significantly increased in urine from subjects consuming processed red meat (i.e. 1-Methylhistidine levels), or unprocessed white meat (i.e. 3-Methylhistidine). ATNC levels significantly increased during the high drinking water nitrate period, which was more pronounced in the processed red meat group. Genotoxicity in Caco-2 cells exposed to faecal water resulted in increased genotoxicity after the interventions, but results were only significant in the low drinking water nitrate period in subjects consuming processed red meat. Furthermore, a positive correlation was found between the ratio of nitrate/vitamin C intake (including drinking water) and the level of ATNC in faecal water of subjects in the processed red meat group, but this was not statistically significant. CONCLUSIONS: Drinking water nitrate significantly contributed to the endogenous formation of NOC, independent of the meat type consumed. This implies that drinking water nitrate levels should be taken into account when evaluating the effect of meat consumption on endogenous formation of NOC. TRIAL REGISTRATION: Dutch Trialregister: 29707 . Registered 19th of October 2018. Retrospectively registered.

Consumption of Nitrate-Rich Beetroot Juice with or without Vitamin C Supplementation Increases the Excretion of Urinary Nitrate, Nitrite, and N-nitroso Compounds in Humans.

Consumption of nitrate-rich beetroot juice (BRJ) by athletes induces a number of beneficial physiological health effects, which are linked to the formation of nitric oxide (NO) from nitrate. However, following a secondary pathway, NO may also lead to the formation of N-nitroso compounds (NOCs), which are known to be carcinogenic in 39 animal species. The extent of the formation of NOCs is modulated by various other dietary factors, such as vitamin C. The present study investigates the endogenous formation of NOCs after BRJ intake and the impact of vitamin C on urinary NOC excretion. In a randomized, controlled trial, 29 healthy recreationally active volunteers ingested BRJ with or without additional vitamin C supplements for one week. A significant increase of urinary apparent total N-nitroso Compounds (ATNC) was found after one dose (5 to 47 nmol/mmol: p < 0.0001) and a further increase was found after seven consecutive doses of BRJ (104 nmol/mmol: p < 0.0001). Vitamin C supplementation inhibited ATNC increase after one dose (16 compared to 72 nmol/mmol, p < 0.01), but not after seven daily doses. This is the first study that shows that BRJ supplementation leads to an increase in formation of potentially carcinogenic NOCs. In order to protect athlete's health, it is therefore important to be cautious with chronic use of BRJ to enhance sports performances.

Persistence of Epigenomic Effects After Recovery From Repeated Treatment With Two Nephrocarcinogens.

The discovery of the epigenetic regulation of transcription has provided a new source of mechanistic understanding to long lasting effects of chemicals. However, this information is still seldom exploited in a toxicological context and studies of chemical effect after washout remain rare. Here we studied the effects of two nephrocarcinogens on the human proximal tubule cell line RPTEC/TERT1 using high-content mRNA microarrays coupled with miRNA, histone acetylation (HA) and DNA methylation (DM) arrays and metabolomics during a 5-day repeat-dose exposure and 3 days after washout. The mycotoxin ochratoxin A (OTA) was chosen as a model compound for its known impact on HA and DM. The foremost effect observed was the modulation of thousands of mRNAs and histones by OTA during and after exposure. In comparison, the oxidant potassium bromate (KBrO3) had a milder impact on gene expression and epigenetics. However, there was no strong correlation between epigenetic modifications and mRNA changes with OTA while with KBrO3 the gene expression data correlated better with HA for both up- and down-regulated genes. Even when focusing on the genes with persistent epigenetic modifications after washout, only half were coupled to matching changes in gene expression induced by OTA, suggesting that while OTA causes a major effect on the two epigenetic mechanisms studied, these alone cannot explain its impact on gene expression. Mechanistic analysis confirmed the known activation of Nrf2 and p53 by KBrO3, while OTA inhibited most of the same genes, and genes involved in the unfolded protein response. A few miRNAs could be linked to these effects of OTA, albeit without clear contribution of epigenetics to the modulation of the pathways at large. Metabolomics revealed disturbances in amino acid balance, energy catabolism, nucleotide metabolism and polyamine metabolism with both chemicals. In conclusion, the large impact of OTA on transcription was confirmed at the mRNA level but also with two high-content epigenomic methodologies. Transcriptomic data confirmed the previously reported activation (by KBrO3) and inhibition (by OTA) of protective pathways. However, the integration of omic datasets suggested that HA and DM were not driving forces in the gene expression changes induced by either chemical.

Drinking Water Nitrate and Human Health: An Updated Review.

Nitrate levels in our water resources have increased in many areas of the world largely due to applications of inorganic fertilizer and animal manure in agricultural areas. The regulatory limit for nitrate in public drinking water supplies was set to protect against infant methemoglobinemia, but other health effects were not considered. Risk of specific cancers and birth defects may be increased when nitrate is ingested under conditions that increase formation of N-nitroso compounds. We previously reviewed epidemiologic studies before 2005 of nitrate intake from drinking water and cancer, adverse reproductive outcomes and other health effects. Since that review, more than 30 epidemiologic studies have evaluated drinking water nitrate and these outcomes. The most common endpoints studied were colorectal cancer, bladder, and breast cancer (three studies each), and thyroid disease (four studies). Considering all studies, the strongest evidence for a relationship between drinking water nitrate ingestion and adverse health outcomes (besides methemoglobinemia) is for colorectal cancer, thyroid disease, and neural tube defects. Many studies observed increased risk with ingestion of water nitrate levels that were below regulatory limits. Future studies of these and other health outcomes should include improved exposure assessment and accurate characterization of individual factors that affect endogenous nitrosation.

Integrated 'omics analysis reveals new drug-induced mitochondrial perturbations in human hepatocytes.

We performed a multiple 'omics study by integrating data on epigenomic, transcriptomic, and proteomic perturbations associated with mitochondrial dysfunction in primary human hepatocytes caused by the liver toxicant valproic acid (VPA), to deeper understand downstream events following epigenetic alterations in the mitochondrial genome. Furthermore, we investigated persistence of cross-omics changes after terminating drug treatment. Upon transient methylation changes of mitochondrial genes during VPA-treatment, increasing complexities of gene-interaction networks across time were demonstrated, which normalized during washout. Furthermore, co-expression between genes and their corresponding proteins increased across time. Additionally, in relation to persistently decreased ATP production, we observed decreased expression of mitochondrial complex I and III-V genes. Persistent transcripts and proteins were related to citric acid cycle and ß-oxidation. In particular, we identified a potential novel mitochondrial-nuclear signaling axis, MT-CO2-FN1-MYC-CPT1. In summary, this cross-omics study revealed dynamic responses of the mitochondrial epigenome to an impulse toxicant challenge resulting in persistent mitochondrial dysfunctioning. Moreover, this approach allowed for discriminating between the toxic effect of VPA and adaptation.

A cross-omics approach to investigate temporal gene expression regulation by 5-hydroxymethylcytosine via TBH-derived oxidative stress showed involvement of different regulatory kinases.

Regulation of DNA methylation plays a crucial role in biological processes and carcinogenesis. The formation of 5-hydroxymethylcytosine (5hmC) by oxidation of 5-methylcytosine (5mC) has been proposed as an intermediate of active demethylation. However, whether and how active demethylation is regulated by oxidative stress-related processes is not well understood. Here we investigated whether free oxygen radicals are capable of directly forming 5hmC and how this enhanced whole genome gene expression. We applied LC-MS/MS technology for the analysis of 5mC, 5hmC, 5-formylcytosine (5fC) and 5-hydroxymethyluracyl (5hmU) in HepG2 cells exposed to hydroxyl- and methyl radicals, formed by tert-butyl hydroperoxide (TBH) at multiple time points. We observed that TBH is able to induce a significant increase in 5hmC. A detailed evaluation of the hydroxymethylome using a combination of 5hmC-immunoprecipitation and microarrays resulted in the identification of highly dynamic modifications that appear to increase during prolonged oxidant exposure. Analyses of temporal gene expression changes in combination with network analysis revealed different subnetworks containing differentially expressed genes (DEGs) with differentially hydroxyl-methylated regions (DhMRs) in different regulatory kinases enriched with serine-threonine kinases. These serine-threonine kinases compromises MAPK14, RPSK6KA1, RIPK1, and PLK3 and were all previously identified as key-regulators in hepatocarcinogenesis and subject of study for chemotherapeutic interventions.

Smart Combinations of Bioactive Compounds in Fruits and Vegetables May Guide New Strategies for Personalized Prevention of Chronic Diseases.

There is ample scientific evidence suggesting that the health benefits of eating the right amounts of a variety of vegetables and fruit are the consequence of the combined action of different phytochemicals. The present review provides an update of the scientific literature on additive and synergistic effects of mixtures of phytochemicals. Most research has been carried out in in vitro systems in which synergistic or additive effects have been established on the level of cell proliferation, apoptosis, antioxidant capacity, and tumor incidence, accompanied by changes in gene and protein expression in relevant pathways underlying molecular mechanisms of disease prevention. The number of human dietary intervention studies investigating complex mixtures of phytochemicals is relatively small, but showing promising results. These studies have demonstrated that combining transcriptomic data with phenotypic markers provide insight into the relevant cellular processes which contribute to the antioxidant response of complex mixtures of phytochemicals. Future studies should be designed as short-term studies testing different combinations of vegetables and fruit, in which markers for disease outcome as well as molecular ('omics)-markers and genetic variability between subjects are included. This will create new opportunities for food innovation and the development of more personalized strategies for prevention of chronic diseases.

Integrative omics data analyses of repeated dose toxicity of valproic acid in vitro reveal new mechanisms of steatosis induction.

Valproic acid (VPA) is a very potent anti-cancer and neuro-protective drug probably by its HDAC inhibiting properties, which may cause steatosis in the liver. The present study investigates the effect of repetitive VPA treatment of primary human hepatocytes (PHH) on whole genome gene expression-, DNA methylation-, and miRNA changes, using microarrays and integrated data analyses. PHH were exposed to a non-cytotoxic dose of VPA for 5days daily which induced lipid accumulation. Part of the PHH was left untreated for 3days for studying the persistence of 'omics' changes. VPA treatment appeared to inhibit the expression of the transcription factors HNF1A and ONECUT1. HNF1A interacted with 41 differentially expressed genes of which 12 were also differentially methylated. None of the genes present in this network were regulated by a DE-miR. The subnetwork of ONECUT1 consisted of 44 differentially expressed genes of which 15 were differentially methylated, and 3 were regulated by a DE-miR. A number of genes in the networks are involved in fatty acid metabolism, and may contribute to the development of steatosis by increasing oxidative stress thereby causing mitochondrial dysfunction, and by shifting metabolism of VPA towards ß-oxidation due to reduced glucuronidation. Part of the changes remained persistent after washing out of VPA, like PMAIP1 which is associated with cellular stress in liver of patients with NASH. The MMP2 gene showed the highest number of interactions with other persistently expressed genes, among which LCN2 which is a key modulator of lipid homeostasis. Furthermore, VPA modulated the expression and DNA methylation level of nuclear receptors and their target genes involved in the adverse outcome pathway of steatosis, thereby expanding our current knowledge of the pathway. In particular, VPA modulated PPARγ, and PPARα, AHR and CD36 on both the gene expression and the DNA methylation level, thereby inhibiting ß-oxidation and increasing uptake of fatty acid into the hepatocytes, respectively. Overall, our integrative data analyses identified novel genes modulated by VPA, which provide more insight into the mechanisms of repeated dose toxicity of VPA, leading to steatosis.

Improved Preventive Effects of Combined Bioactive Compounds Present in Different Blueberry Varieties as Compared to Single Phytochemicals.

Blueberries contain many different phytochemicals which might be responsible for their disease preventive properties. In a previously conducted human dietary intervention study, we showed that a 4-week intervention with blueberry⁻apple juice protected the participants against oxidative stress and modulated expression of genes involved in different genetic pathways contributing to the antioxidant response. The present study investigates the effect of different blueberry varieties (Elliot, Draper, Bluecrop, and Aurora, and the blueberry⁻apple juice from our previous human dietary intervention study), and four different single compounds (vitamin C, peonidin, cyanidin, and quercetin) on antioxidant capacity and gene expression changes in colonic cells in vitro, and compares the outcome with the earlier in vivo findings. The results demonstrate that all blueberry varieties as well as the blueberry⁻apple juice were more effective in reducing oxidative stress as compared to the single compounds (e.g., DNA strand break reduction: EC50: Elliot 8.3 mg/mL, Aurora and Draper 11.9 mg/mL, blueberry⁻apple juice 12.3 mg/mL, and Bluecrop 12.7 mg/mL; single compounds). In addition, the gene expression profiles (consisting of 18 selected genes from the in vivo study) induced by the blueberry varieties were more similar to the profile of the human intervention study (range 44⁻78%). The blueberry variety Elliot showed the strongest and most similar effects, almost 80% of gene expression modulations were similar compared to the in vivo results. From the single compounds (range 17⁻44%), quercetin induced the most comparable gene expression changes, i.e., 44%. This approach could be useful in agriculture for identifying crop varieties containing combinations of phytochemicals which show optimal preventive capacities.

Nuclear and Mitochondrial DNA Methylation Patterns Induced by Valproic Acid in Human Hepatocytes.

Valproic acid (VPA) is one of the most widely prescribed antiepileptic drugs in the world. Despite its pharmacological importance, it may cause liver toxicity and steatosis through mitochondrial dysfunction. The aim of this study is to further investigate VPA-induced mechanisms of steatosis by analyzing changes in patterns of methylation in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, primary human hepatocytes (PHHs) were exposed to an incubation concentration of VPA that was shown to cause steatosis without inducing overt cytotoxicity. VPA was administered daily for 5 days, and this was followed by a 3 day washout (WO). Methylated DNA regions (DMRs) were identified by using the methylated DNA immunoprecipitation-sequencing (MeDIP-seq) method. The nDNA DMRs after VPA treatment could indeed be classified into oxidative stress- and steatosis-related pathways. In particular, networks of the steatosis-related gene EP300 provided novel insight into the mechanisms of toxicity induced by VPA treatment. Furthermore, we suggest that VPA induces a crosstalk between nDNA hypermethylation and mtDNA hypomethylation that plays a role in oxidative stress and steatosis development. Although most VPA-induced methylation patterns appeared reversible upon terminating VPA treatment, 31 nDNA DMRs (including 5 zinc finger protein genes) remained persistent after the WO period. Overall, we have shown that MeDIP-seq analysis is highly informative in disclosing novel mechanisms of VPA-induced toxicity in PHHs. Our results thus provide a prototype for the novel generation of interesting methylation biomarkers for repeated dose liver toxicity in vitro.

Integrative "-Omics" Analysis in Primary Human Hepatocytes Unravels Persistent Mechanisms of Cyclosporine A-Induced Cholestasis.

Cyclosporine A (CsA) is an undecapeptide with strong immunosuppressant activities and is used a lot after organ transplantation. Furthermore, it may induce cholestasis in the liver. In general, the drug-induced cholestasis (DIC) pathway includes genes involved in the uptake, synthesis, conjugation, and secretion of bile acids. However, whether CsA-induced changes in the cholestasis pathway in vitro are persistent for repeated dose toxicity has not yet been investigated. To explore this, primary human hepatocytes (PHH) were exposed to a subcytotoxic dose of 30 µM CsA daily for 3 and 5 days. To investigate the persistence of induced changes upon terminating CsA exposure after 5 days, a subset of PHH was subjected to a washout period (WO-period) of 3 days. Multiple -omics analyses, comprising whole genome analysis of DNA methylation, gene expression, and microRNA expression, were performed. The CsA-treatment resulted after 3 and 5 days, respectively, in 476 and 20 differentially methylated genes (DMGs), 1353 and 1481 differentially expressed genes (DEGs), and in 22 and 29 differentially expressed microRNAs (DE-miRs). Cholestasis-related pathways appeared induced during CsA-treatment. Interestingly, 828 persistent DEGs and 6 persistent DE-miRs but no persistent DMGs were found after the WO-period. These persistent DEGs and DE-miRs showed concordance for 22 genes. Furthermore, 29 persistent DEGs changed into the same direction as observed in livers from cholestasis patients. None of those 29 DEGs which among others relate to oxidative stress and lipid metabolism are yet present in the DIC pathway or cholestasis adverse outcome pathway (AOP) thus presenting novel findings. In summary, we have demonstrated for the first time a persistent impact of repeated dose administration of CsA on genes and microRNAs related to DIC in the gold standard human liver in vitro model with PHH.