Coordinated Regulation of UGT2B15 Expression by Long Noncoding RNA LINC00574 and hsa-miR-129-5p in HepaRG Cells.

Recent studies have shown that microRNAs and long noncoding RNAs (lncRNAs) regulate the expression of drug metabolizing enzymes (DMEs) in human hepatic cells and that a set of DMEs, including UDP glucuronosyltransferase (UGT) 2B15, is down-regulated dramatically in liver cells by toxic acetaminophen (APAP) concentrations. In this study we analyzed mRNA, microRNA, and lncRNA expression profiles in APAP-treated HepaRG cells to explore noncoding RNA-dependent regulation of DME expression. The expression of UGT2B15 and lncRNA LINC00574 was decreased in APAP-treated HepaRG cells. UGT2B15 levels were diminished by LINC00574 suppression using antisense oligonucleotides or small interfering RNA. Furthermore, we found that hsa-miR-129-5p suppressed LINC00574 and decreased UGT2B15 expression via LINC00574 in HepaRG cells. In conclusion, our results indicate that LINC00574 acts as an important regulator of UGT2B15 expression in human hepatic cells, providing experimental evidence and new clues to understand the role of cross-talk between noncoding RNAs. SIGNIFICANCE STATEMENT: We showed a molecular network that displays the cross-talk and consequences among mRNA, micro RNA, long noncoding RNA, and proteins in acetaminophen (APAP)-treated HepaRG cells. APAP treatment increased the level of hsa-miR-129-5p and decreased that of LINC00574, ultimately decreasing the production of UDP glucuronosyltransferase (UGT) 2B15. The proposed regulatory network suppresses UGT2B15 expression through interaction of hsa-miR-129-5p and LINC00574, which may be achieved potentially by recruiting RNA binding proteins.

Long noncoding RNA LINC00844-mediated molecular network regulates expression of drug metabolizing enzymes and nuclear receptors in human liver cells.

Noncoding RNAs, such as long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), regulate gene expression in many physiological and pathological processes, including drug metabolism. Drug metabolizing enzymes (DMEs) are critical components in drug-induced liver toxicity. In this study, we used human hepatic HepaRG cells treated with 5 or 10 mM acetaminophen (APAP) as a model system and identified LINC00844 as a toxicity-responsive lncRNA. We analyzed the expression profiles of LINC00844 in different human tissues. In addition, we examined the correlations between the levels of LINC00844 and those of key DMEs and nuclear receptors (NRs) for APAP metabolism in humans. Our results showed that lncRNA LINC00844 is enriched in the liver and its expression correlates positively with mRNA levels of CYP3A4, CYP2E1, SULT2A1, pregnane X receptor (PXR), and hepatocyte nuclear factor (HNF) 4α. We demonstrated that LINC00844 regulates the expression of these five genes in HepaRG cells using gain- and loss-of-function assays. Further, we discovered that LINC00844 is localized predominantly in the cytoplasm and acts as an hsa-miR-486-5p sponge, via direct binding, to protect SULT2A1 from miRNA-mediated gene silencing. Our data also demonstrated a functional interaction between LINC00844 and hsa-miR-486-5p in regulating DME and NR expression in HepaRG cells and primary human hepatocytes. We depicted a LINC00844-mediated regulatory network that involves miRNA and NRs and influences DME expression in response to APAP toxicity.

Regulation of cytochrome P450 expression by microRNAs and long noncoding RNAs: Epigenetic mechanisms in environmental toxicology and carcinogenesis.

Environmental exposures to hazardous chemicals are associated with a variety of human diseases and disorders, including cancers. Phase I metabolic activation and detoxification reactions catalyzed by cytochrome P450 enzymes (CYPs) affect the toxicities of many xenobiotic compounds. Proper regulation of CYP expression influences their biological effects. Noncoding RNAs (ncRNAs) are involved in regulating CYP expression, and ncRNA expression is regulated in response to environmental chemicals. The mechanistic interactions between ncRNAs and CYPs associated with the toxicity and carcinogenicity of environmental chemicals are described in this review, focusing on microRNA-dependent CYP regulation. The role of long noncoding RNAs in regulating CYP expression is also presented and new avenues of research concerning this regulatory mechanism are described.

Multiple microRNAs function as self-protective modules in acetaminophen-induced hepatotoxicity in humans.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure. Yet the mechanisms underlying adaptive tolerance toward APAP-induced liver injury are not fully understood. To better understand molecular mechanisms contributing to adaptive tolerance to APAP is an underpinning foundation for APAP-related precision medicine. In the current study, the mRNA and microRNA (miRNA) expression profiles derived from next generation sequencing data for APAP-treated (5 and 10 mM) HepaRG cells and controls were analyzed systematically. Putative miRNAs targeting key dysregulated genes involved in APAP hepatotoxicity were selected using in silico prediction algorithms, un-biased gene ontology, and network analyses. Luciferase reporter assays, RNA electrophoresis mobility shift assays, and miRNA pull-down assays were performed to investigate the role of miRNAs affecting the expression of dysregulated genes. Levels of selected miRNAs were measured in serum samples obtained from children with APAP overdose (58.6-559.4 mg/kg) and from healthy controls. As results, 2758 differentially expressed genes and 47 miRNAs were identified. Four of these miRNAs (hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p) suppressed drug metabolizing enzyme (DME) levels involved in APAP-induced liver injury by downregulating HNF1A, HNF4A and NR1I2 expression. Exogenous transfection of these miRNAs into HepaRG cells effectively rescued them from APAP toxicity, as indicated by decreased alanine aminotransferase levels. Importantly, hsa-miR-320a and hsa-miR-877-5p levels were significantly elevated in serum samples obtained from children with APAP overdose compared to health controls. Collectively, these data indicate that hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p suppress DME expression involved in APAP-induced hepatotoxicity and they contribute to an adaptive response in hepatocytes.

Activation of the Nrf2 signaling pathway in usnic acid-induced toxicity in HepG2 cells.

Many usnic acid-containing dietary supplements have been marketed as weight loss agents, although severe hepatotoxicity and acute liver failure have been associated with their overuse. Our previous mechanistic studies revealed that autophagy, disturbance of calcium homeostasis, and ER stress are involved in usnic acid-induced toxicity. In this study, we investigated the role of oxidative stress and the Nrf2 signaling pathway in usnic acid-induced toxicity in HepG2 cells. We found that a 24-h treatment with usnic acid caused DNA damage and S-phase cell cycle arrest in a concentration-dependent manner. Usnic acid also triggered oxidative stress as demonstrated by increased reactive oxygen species generation and glutathione depletion. Short-term treatment (6 h) with usnic acid significantly increased the protein level for Nrf2 (nuclear factor erythroid 2-related factor 2), promoted Nrf2 translocation to the nucleus, up-regulated antioxidant response element (ARE)-luciferase reporter activity, and induced the expression of Nrf2-regulated targets, including glutathione reductase, glutathione S-transferase, and NAD(P)H quinone oxidoreductase-1 (NQO1). Furthermore, knockdown of Nrf2 with shRNA potentiated usnic acid-induced DNA damage and cytotoxicity. Taken together, our results show that usnic acid causes cell cycle dysregulation, DNA damage, and oxidative stress and that the Nrf2 signaling pathway is activated in usnic acid-induced cytotoxicity.

Metabolic activation of pyrrolizidine alkaloids leading to phototoxicity and photogenotoxicity in human HaCaT keratinocytes.

Pyrrolizidine alkaloids, produced by a large number of poisonous plants with wide global distribution, are associated with genotoxicity, tumorigenicity, and hepatotoxicity in animals and humans. Mammalian metabolism converts pyrrolizidine alkaloids to reactive pyrrolic metabolites (dehydropyrrolizidine alkaloids) that form covalent protein and DNA adducts. Although a mechanistic understanding is currently unclear, pyrrolizidine alkaloids can cause secondary (hepatogenous) photosensitization and induce skin cancer. In this study, the phototoxicity of monocrotaline, riddelliine, dehydromonocrotaline, dehydroriddelliine, and dehydroretronecine (DHR) in human HaCaT keratinocytes under ultraviolet A (UVA) irradiation was determined. UVA irradiation of HaCaT cells treated with dehydromonocrotaline, dehydroriddelline, and DHR resulted in increased release of lactate dehydrogenase and enhanced photocytotoxicity proportional to the UVA doses. UVA-induced photochemical DNA damage also increased proportionally with dehydromonocrotaline and dehydroriddelline. UVA treatment potentiated the formation of 8-hydroxy-2'-deoxyguanosine DNA adducts induced by dehydromonocrotaline in HaCaT skin keratinocytes. Using electron spin resistance trapping, we found that UVA irradiation of dehydromonocrotaline and dehydroriddelliine generates reactive oxygen species (ROS), including hydroxyl radical, singlet oxygen, and superoxide, and electron transfer reactions, indicating that cytotoxicity and genotoxicity of these compounds could be mediated by ROS. Our results suggest that dehydropyrrolizidine alkaloids formed or delivered to the skin cause pyrrolizidine alkaloid-induced secondary photosensitization and possible skin cancer.

Toxicogenomics and cancer susceptibility: advances with next-generation sequencing.

The aim of this review is to comprehensively summarize the recent achievements in the field of toxicogenomics and cancer research regarding genetic-environmental interactions in carcinogenesis and detection of genetic aberrations in cancer genomes by next-generation sequencing technology. Cancer is primarily a genetic disease in which genetic factors and environmental stimuli interact to cause genetic and epigenetic aberrations in human cells. Mutations in the germline act as either high-penetrance alleles that strongly increase the risk of cancer development, or as low-penetrance alleles that mildly change an individual's susceptibility to cancer. Somatic mutations, resulting from either DNA damage induced by exposure to environmental mutagens or from spontaneous errors in DNA replication or repair are involved in the development or progression of the cancer. Induced or spontaneous changes in the epigenome may also drive carcinogenesis. Advances in next-generation sequencing technology provide us opportunities to accurately, economically, and rapidly identify genetic variants, somatic mutations, gene expression profiles, and epigenetic alterations with single-base resolution. Whole genome sequencing, whole exome sequencing, and RNA sequencing of paired cancer and adjacent normal tissue present a comprehensive picture of the cancer genome. These new findings should benefit public health by providing insights in understanding cancer biology, and in improving cancer diagnosis and therapy.

Microcalorimetric Investigations of Reversible Staphylococcal Enterotoxin Unfolding.

Staphylococcal food poisoning (SFP) is a common food-borne illness often associated with contamination during food handling. The genes for Staphylococcal enterotoxin (SE) isoforms SEA and SEB are frequently detected in human nasal Staphylococcus aureus isolates and these toxins are commonly associated with SFP. Past studies described the resistance of preformed SE proteins to heat inactivation and their reactivation upon cooling in foods. Full thermodynamic analyses for these processes have not been reported, however. The thermal stabilities of SEA, SEB, and SEH and reversibility of unfolding in simple buffers were investigated at pH 4.5 and pH 6.8 using differential scanning calorimetry (DSC). SEA and SEB unfolding was irreversible at pH 6.8 and at least partially reversible at pH 4.5 while SEH unfolding was irreversible at pH 4.5 and reversible at pH 6.8. Additional studies showed maximum refolding for SEB at pH 3.5-4.0 and diminished refolding at pH 4.5 with increasing ionic strength. SE-stimulated secretion of interferon-gamma by human peripheral blood mononuclear cells was used to assess residual SE biological activity following heat treatments using conditions matching those used for DSC studies. The biological activities of SEB and SEH exhibited greater resistance to heat inactivation than that of SEA. The residual activities of heat-treated SEB and SEH were measurable but diminished further in the presence of reconstituted nonfat dry milk adjusted to pH 4.5 or pH 6.8. To different extents, the pH and ionic strengths typical for foods influenced the thermal stabilities of SEA, SEB, and SEH and their potentials to renature spontaneously after heat treatments.

Characterization of cytochrome P450s (CYP)-overexpressing HepG2 cells for assessing drug and chemical-induced liver toxicity.

Hepatic metabolism catalyzed by the cytochrome P450 (CYP) superfamily affects liver toxicity associated with exposures to natural compounds and xenobiotic agents. Previously we generated a battery of HepG2-derived stable cell lines that individually express 14 CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7). In this study, we comprehensively characterized each cell line for its CYP expression and enzyme activity. Specifically, we measured the mRNA expression, protein expression, and metabolite formation. Using CYP3A4, 2D6, and 2C9-overexpressing cells as representatives, we examined the stability of these cells in long-term cultures for up to 10 passages. The results showed that CYPs can be stably overexpressed for up to 10 cell culture passages without losing their activities. The robustness of responses to stimuli among the cells at different passages was also investigated in CYP3A4-overexpressing cells and the response to amiodarone and dronedarone showed no difference between the cells at the passage 2 and 10. Moreover, the mRNA expression level of most CYPs was higher in CYP-overexpressing HepG2 cells than that in HepaRG cells and primary human hepatocytes. This study confirmed the stability of CYP-overexpressing HepG2 cell lines and provided useful information for a broader use of these cells in pharmacologic and toxicologic research.

A functional quantitative polymerase chain reaction assay for ricin, Shiga toxin, and related ribosome-inactivating proteins.

The potent toxins ricin, abrin, and other ribosome-inactivating proteins deadenylate a specific base in 28S ribosomal RNA that destroys ribosomes and leads to cell death. We have taken advantage of the fact that reverse transcriptase preferentially inserts an adenine opposite to an abasic site in RNA to create a quantitative polymerase chain reaction (PCR) assay to detect the damage. This assay detects as little as 30pg of ricin. We used the assay to study enzymatic properties of ricin such as pH and temperature optima (pH 4.5-5.0 and 60 degrees C).

Immunomagnetic Capture of Big Six Shiga Toxin-Producing Escherichia coli Strains in Apple Juice with Detection by Multiplex Real-Time PCR Eliminates Interference from the Food Matrix.

Having reliable methods for detecting Shiga toxin-producing Escherichia coli (STEC) in foods is an important food safety goal. The majority of STEC outbreaks have involved either the O157:H7 serotype or one of six non-O157 serogroups, O26, O45, O103, O111, O121, and O145, termed "The Big Six." We have compared detection by PCR of the Shiga toxin genes stx1a and stx2a from STEC bacteria isolated from unclarified apple juice by simple centrifugation with the use of an immunocapture technique to minimize contaminants (such as pectin and polyphenols that may copurify with DNA) that may interfere with DNA amplification efficiencies and limit sensitivity. An internal control for successful immunocapture, DNA extraction, and PCR amplification was generated by introducing the pmRaspberry plasmid into an stx null strain, yielding an E. coli O45 pmRaspberry derivative that can be added to food samples directly. Using serial dilutions of a representative Big Six STEC in apple juice, our immunocapture method resulted in a 50% probability of detection value of 3.34, 2.25, and 4.25 CFU for detection by multiplex real-time PCR, growth on solid agar, and multiplex endpoint PCR, respectively. The time to result was 6.5 h, 9.5 h, and 1.5 days for immunocapture of Big Six STECs and detection by multiplex real-time PCR, endpoint PCR, and growth on solid agar, respectively. A set of 52 Big Six STEC isolates and 30 non-Big Six STEC strains was used to establish the inclusivity and exclusivity of the method. Finally, the ability to detect Big Six STEC contamination reliably was confirmed at 4.5 and 45 CFU/25-mL portions of refrigerated apple juice.

Primary and secondary pyrrolic metabolites of pyrrolizidine alkaloids form DNA adducts in human A549 cells.

Humans and animals can be exposed to carcinogenic pyrrolizidine alkaloids (PAs) through consumption of plants commonly found in many parts of the world. Although the liver is the primary target organ for carcinogenic PAs, they have also induced lung tumors in rodents. Hepatic cytochrome P450 activity converts PAs into dehydro-PAs that can be hydrolyzed to dehydropyrrolizidine (DHP); these reactive pyrrolic metabolites can produce four characteristic DNA adducts associated with PA-induced liver tumor initiation in laboratory animals. We reported recently that these four DNA adducts are also formed when 7-glutathione-DHP (7-GS-DHP) or 7-cysteine-DHP is incubated with calf thymus DNA. Here we showed that the four characteristic DNA adducts were formed when human A549 brochoalveolar carcinoma cells were treated with three dehydro-PAs (dehydroriddelliine, dehydromonocrotaline, or dehydroretronecine) or with 7-GS-DHP or 7-cysteine-DHP. For comparison, two parent PAs (riddelliine and monocrotaline) and 7,9-di-glutathionine-DHP were studied. No DHP-DNA adducts were detected with these incubations, confirming that A549 lung carcinoma cells do not express cytochrome P450 enzymes required for metabolic activation of PAs. Our results show that primary and secondary pyrrolic metabolites of carcinogenic PAs produce characteristic DHP-containing DNA adducts in A549 lung cancer cells, suggesting that they are DNA reactive metabolites.

MicroRNAs hsa-miR-495-3p and hsa-miR-486-5p suppress basal and rifampicin-induced expression of human sulfotransferase 2A1 (SULT2A1) by facilitating mRNA degradation.

Drug metabolizing enzymes mediate biotransformation of drugs and play an essential role in drug efficacy and toxicity. Human sulfotransferases are a superfamily of Phase II detoxification enzymes that metabolize a wide spectrum of endogenous compounds and xenobiotics. SULT2A1 is one of the most abundant hepatic sulfotransferases and it catalyzes the sulfate conjugation of many endogenous substrates, such as bile acids and steroids. In the current study, we utilized a systematic approach by combining a series of computational analyses and in vitro methods to identify miRNAs that repress SULT2A1 expression post-transcriptionally. Our in silico analyses predicted miRNA response elements for hsa-miR-495-3p and hsa-miR-486-5p within the 3'-UTR of SULT2A1 mRNA and the levels of these miRNAs were inversely correlated with that of SULT2A1 mRNA in human liver. Using fluorescence-based RNA electrophoretic mobility shift assays, we found that hsa-miR-495-3p and hsa-miR-486-5p interacted directly with the SULT2A1 3'-UTR. The activity of a luciferase reporter gene construct containing sequences from the SULT2A1 3-UTR was suppressed by hsa-miR-486-5p and hsa-miR-495-3p. Furthermore, gain- and loss-of-function assays demonstrated that hsa-miR-486-5p and hsa-miR-495-3p negatively modulate basal and rifampicin-induced expression of SULT2A1 in HepG2 cells by decreasing mRNA stability.

Effect of p53 genotype on gene expression profiles in murine liver.

The tumor suppressor protein p53 is a key regulatory element in the cell and is regarded as the "guardian of the genome". Much of the present knowledge of p53 function has come from studies of transgenic mice in which the p53 gene has undergone a targeted deletion. In order to provide additional insight into the impact on the cellular regulatory networks associated with the loss of this gene, microarray technology was utilized to assess gene expression in tissues from both the p53(-/-) and p53(+/-) mice. Six male mice from each genotype (p53(+/+), p53(+/-), and p53(-/-)) were humanely killed and the tissues processed for microarray analysis. The initial studies have been performed in the liver for which the Dunnett test revealed 1406 genes to be differentially expressed between p53(+/+) and p53(+/-) or between p53(+/+) and p53(-/-) at the level of p < or = 0.05. Both genes with increased expression and decreased expression were identified in p53(+/-) and in p53(-/-) mice. Most notable in the gene list derived from the p53(+/-) mice was the significant reduction in p53 mRNA. In the p53(-/-) mice, not only was there reduced expression of the p53 genes on the array, but genes associated with DNA repair, apoptosis, and cell proliferation were differentially expressed, as expected. However, altered expression was noted for many genes in the Cdc42-GTPase pathways that influence cell proliferation. This may indicate that alternate pathways are brought into play in the unperturbed liver when loss or reduction in p53 levels occurs.

Mitochondrial dysfunction induced by leflunomide and its active metabolite.

Leflunomide, an anti-inflammatory drug used for the treatment of rheumatoid arthritis, has been marked with a black box warning regarding an increased risk of liver injury. The active metabolite of leflunomide, A771726, which also carries a boxed warning about potential hepatotoxicity, has been marketed as teriflunomide for the treatment of relapsing multiple sclerosis. Thus far, however, the mechanism of liver injury associated with the two drugs has remained elusive. In this study, cytotoxicity assays showed that ATP depletion and subsequent LDH release were induced in a time- and concentration-dependent manner by leflunomide in HepG2 cells, and to a lesser extent, by A77 1726. The decline of cellular ATP levels caused by leflunomide was dramatically exacerbated when galactose was substituted for glucose as the sugar source, indicating a potential mitochondrial liability of leflunomide. By measuring the activities of immuno-captured mitochondrial oxidative phosphorylation (OXPHOS) complexes, we found that leflunomide and A77 1726 preferentially targeted complex V (F1FO ATP synthase), with IC50 values of 35.0 and 63.7 µM, respectively. Bongkrekic acid, a mitochondrial permeability transition pore blocker that targets adenine nucleotide translocase, profoundly attenuated mitochondrial membrane depolarization, ATP depletion, and LDH leakage induced by leflunomide and A77 1726. Substantial alterations of mitochondrial function at the transcript level were observed in leflunomide-treated HepG2 cells, whereas the effects of A77 1726 on the cellular transcriptome were much less profound. Our results suggest that mitochondrial dysfunction may be implicated in the hepatotoxicity associated with leflunomide and A77 1726, with the former exhibiting higher toxicity potency.

A systematic evaluation of microRNAs in regulating human hepatic CYP2E1.

Cytochrome P450 2E1 (CYP2E1) is an important drug metabolizing enzyme for processing numerous xenobiotics in the liver, including acetaminophen and ethanol. Previous studies have shown that microRNAs (miRNAs) can suppress CYP2E1 expression by binding to the 3'-untranslated region (3'-UTR) of its transcript. However, a systematic analysis of CYP2E1 regulation by miRNAs has not been described. Here, we applied in silico, in vivo, and in vitro approaches to investigate miRNAs involved in the regulation of CYP2E1. Initially, potential miRNA binding sites in the CYP2E1 mRNA transcript were identified and screened using in silico methods. Next, inverse correlations were found in human liver samples between the expression of CYP2E1 mRNA and the levels of two miRNA species, hsa-miR-214-3p and hsa-miR-942-5p. In a HepG2-derived CYP2E1 over-expression cell model, hsa-miR-214-3p exhibited strong suppression of CYP2E1 expression by targeting the coding region of its mRNA transcript, but hsa-miR-942-5p did not inhibit CYP2E1 levels. Electrophoretic mobility shift assays confirmed that hsa-miR-214-3p recruited other cellular protein factors to form stable complexes with specific sequences present in the CYP2E1 mRNA open reading frame. Transfection of HepaRG cells with hsa-miR-214-3p mimics inhibited expression of the endogenous CYP2E1 gene. Further, hsa-miR-214-3p mimics partially blocked ethanol-dependent increases in CYP2E1 mRNA and protein levels in HepG2 cells and they reduced the release of alanine aminotransferase from CYP2E1-overexpressing HepG2 cells exposed to acetaminophen. These results substantiate the suppressing effect of hsa-miR-214-3p on CYP2E1 expression.

The expression, induction and pharmacological activity of CYP1A2 are post-transcriptionally regulated by microRNA hsa-miR-132-5p.

Cytochrome P450 1A2 (CYP1A2) is one of the most abundant and important drug metabolizing enzymes in human liver. However, little is known about the post-transcriptional regulation of CYP1A2, especially the mechanisms involving microRNAs (miRNAs). This study applied a systematic approach to investigate the post-transcriptional regulation of CYP1A2 by miRNAs. Candidate miRNAs targeting the 3'-untranslated region (3'-UTR) of CYP1A2 were screened in silico, resulting in the selection of sixty-two potential miRNAs for further analysis. The levels of two miRNAs, hsa-miR-132-5p and hsa-miR-221-5p, were inversely correlated with the expression of CYP1A2 mRNA transcripts in normal human liver tissue samples represented in The Cancer Genome Atlas (TCGA) dataset. The interactions between these miRNAs and cognate CYP1A2 mRNA sequences were evaluated using luciferase reporter gene studies and electrophoretic mobility shift assays, by which a direct interaction was confirmed involving hsa-miR-132-5p and a cognate binding site present in the CYP1A2 3'-UTR. Experiments by which hsa-miR-132-5p or random miRNA controls were introduced into HepG2, Huh-7 and HepaRG hepatic cell lines showed that only hsa-miR-132-5p suppressed the endogenous and lansoprazole-induced expression of CYP1A2, at biological activity, protein production, and mRNA transcript levels. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays showed that hsa-miR-132-5p attenuates CYP1A2-mediated, lansoprazole-enhanced, flutamide-induced hepatic cell toxicity. Results from multilayer experiments demonstrate that hsa-miR-132-5p suppresses the expression of CYP1A2 and that this suppression is able to decrease the extent of an adverse drug-drug interaction involving lansoprazole and flutamide.

Thermal inactivation of ricin using infant formula as a food matrix.

Ricin is a potent protein toxin found in the seeds of the castor bean plant, Ricinus communis. Ricin specifically and irreversibly inactivates ribosomes, promoting cell death by inhibiting protein synthesis. It is composed of a ribosome-inactivating enzyme (A-chain) linked to a lectin (B-chain) by a single disulfide bond. Several reports indicate that ricin can be detoxified by thermal treatment; however, the conditions required for inactivation are not well characterized. In addition, little information exists on the thermal stability of ricin added to foods. The objective of this work was to determine the effects of heat treatments on the detection and toxicity of ricin added to milk- and soy-based infant formulas. Reconstituted infant formula powders containing 100 mug of ricin/mL were heated at 60-90 degrees C for up to 5 h. The heat-treated formulas were analyzed by ELISA to determine levels of ricin. The residual cytotoxicity of ricin-containing infant formula after heat treatments was determined using RAW264.7 mouse macrophage cells. The ELISA and the cytotoxicity assay indicated that ricin detection and toxicity decreased with increasing heating times and temperatures. Minimal losses in detection and toxicity were found for ricin heated at 60 degrees C for 2 h. The half-lives of ricin cytoxic activity in a milk-based infant formula at 60, 70, 75, 80, 85, and 90 degrees C were >100, 9.8 +/- 0.5, 5.8 +/- 0.9, 5.1 +/- 0.7, 3.1 +/- 0.4, and 1.8 +/- 0.2 min, respectively; the comparable values for a soy-based infant formula were >100, 16 +/- 1.6, 8.7 +/- 1.2, 6.9 +/- 1.1, 3.0 +/- 0.4, and 2.0 +/- 0.3 min. ELISA detection was a good indicator of the cytotoxicity of heat-treated ricin. The results indicate that ricin is a relatively heat stable protein and may remain toxic under some food processing conditions.