Role of long non-coding RNA in DEET- and fipronil-mediated alteration of transcripts associated with Phase I and Phase II xenobiotic metabolism in human primary hepatocytes.

Human exposure to environmental chemicals both individually and in combination occurs frequently world-wide most often with unknown consequences. Use of molecular approaches to aide in the assessment of risk involved in chemical exposure is a growing field in toxicology. In this study, we examined the impact of two environmental chemicals used in and around homes, the insect repellent DEET (N,N-diethyl-m-toluamide) and the phenylpyrazole insecticide fipronil (fluocyanobenpyrazole) on transcript levels of enzymes potentially involved in xenobiotic metabolism and on long non-coding RNAs (lncRNAs). Primary human hepatocytes were treated with these two chemicals both individually and in combination. Using RNA-Seq, we found that 10 major enzyme categories involved in phase 1 and phase 2 xenobiotic metabolism were significantly (α = 0.05) up- and down-regulated (i.e., 100 µM DEET-19 transcripts, 89% up and 11% down; 10 µM fipronil-52 transcripts, 53% up and 47% down; and 100 µM DEET +10 µM fipronil-69 transcripts, 43% up and 57% down). The altered genes were then mapped to the human genome and their proximity (within 1,000,000 bp) to lncRNAs examined. Unique proximities were discovered between altered lncRNA and altered P450s (CYP) and other enzymes (DEET, 2 CYP; Fipronil, 6 CYP and 15 other; and DEET + fipronil, 7 CYP and 21 other). Many of the altered P450 transcripts were in multiple clusters in the genome with proximal altered lncRNAs, suggesting a regulator function for the lncRNA. At the gene level there was high percent identity for lncRNAs near P450 clusters, but this relationship was not found at the transcript level. The role of these altered lncRNAs associated with xenobiotic induction, human diseases and chemical mixtures is discussed.

Differential Expression Profile of lncRNAs from Primary Human Hepatocytes Following DEET and Fipronil Exposure.

While the synthesis and use of new chemical compounds is at an all-time high, the study of their potential impact on human health is quickly falling behind, and new methods are needed to assess their impact. We chose to examine the effects of two common environmental chemicals, the insect repellent N,N-diethyl-m-toluamide (DEET) and the insecticide fluocyanobenpyrazole (fipronil), on transcript levels of long non-protein coding RNAs (lncRNAs) in primary human hepatocytes using a global RNA-Seq approach. While lncRNAs are believed to play a critical role in numerous important biological processes, many still remain uncharacterized, and their functions and modes of action remain largely unclear, especially in relation to environmental chemicals. RNA-Seq showed that 100 µM DEET significantly increased transcript levels for 2 lncRNAs and lowered transcript levels for 18 lncRNAs, while fipronil at 10 µM increased transcript levels for 76 lncRNAs and decreased levels for 193 lncRNAs. A mixture of 100 µM DEET and 10 µM fipronil increased transcript levels for 75 lncRNAs and lowered transcript levels for 258 lncRNAs. This indicates a more-than-additive effect on lncRNA transcript expression when the two chemicals were presented in combination versus each chemical alone. Differentially expressed lncRNA genes were mapped to chromosomes, analyzed by proximity to neighboring protein-coding genes, and functionally characterized via gene ontology and molecular mapping algorithms. While further testing is required to assess the organismal impact of changes in transcript levels, this initial analysis links several of the dysregulated lncRNAs to processes and pathways critical to proper cellular function, such as the innate and adaptive immune response and the p53 signaling pathway.

Impact of Environmental Chemicals on the Transcriptome of Primary Human Hepatocytes: Potential for Health Effects.

New paradigms for human health risk assessment of environmental chemicals emphasize the use of molecular methods and human-derived cell lines. In this study, we examined the effects of the insect repellent DEET (N,N-diethyl-m-toluamide) and the phenylpyrazole insecticide fipronil (fluocyanobenpyrazole) on transcript levels in primary human hepatocytes. These chemicals were tested individually and as a mixture. RNA-Seq showed that 100 µM DEET significantly increased transcript levels (α = 0.05) for 108 genes and lowered transcript levels for 64 genes and fipronil at 10 µM increased the levels of 2246 transcripts and decreased the levels for 1428 transcripts. Fipronil was 21-times more effective than DEET in eliciting changes, even though the treatment concentration was 10-fold lower for fipronil versus DEET. The mixture of DEET and fipronil produced a more than additive effect (levels increased for 3017 transcripts and decreased for 2087 transcripts). The transcripts affected for all chemical treatments were classified by GO analysis and mapped to chromosomes. The overall treatment responses, specific pathways, and individual transcripts affected were discussed at different levels of fold-change. Changes found in transcript levels in response to treatments will require further research to understand their importance in overall cellular, organ, and organismic function.

Human variation and risk assessment: microarray and other studies utilizing human hepatocytes and human liver subcellular preparations.

The new paradigms proposed for human health risk assessment stress the need for the use of human and human-derived cell lines, and this review summarizes the use of primary human hepatocytes and hepatocyte subcellular preparations for the investigation of the metabolism and metabolic interactions of environmental chemicals. This includes interactions based on inhibition, induction, and activation. The role of cytotoxicity is also considered. The use of hepatocytes and hepatocyte preparations provides especially important information for the investigation of human variation and is summarized. This area is, at present, relatively neglected but will in the future be essential for accurate assessment of human health risk. A detailed summary of an initial attempt to utilize microarray technology for the study of genome-wide effects is included.

Human environments: definition, scope, and the role of toxicology.

This chapter is a brief introduction to the subject matter of the volume including the complexity and definition of human environments. Exposure to complex mixtures and the problem of interactions are considered as well as the important role of toxicology in environmental and human health, including risk analysis, risk management, and risk communication.

The future of human health risk assessment of environmental chemicals.

Dramatic changes are beginning to take place in risk assessment, changes that will involve rapid, high-throughput assays capable of analyzing large numbers of samples in a short time. These new systems will incorporate many of the aspects of cellular and molecular biology, including human and human-derived cell lines, toxicogenomics, proteomics, and metabolomics. The large amount of data generated will require a systems biology approach if it is to be reduced to generalizations of value in risk management. The current chapter briefly describes the emerging systems and their component parts.

Metabolic interactions of environmental toxicants in humans.

A description of the interactions between environmental toxicants following simultaneous exposure or exposure in close temporal sequence is presented. At the metabolic level, such interactions may be based on induction, inhibition, or activation of xenobiotic-metabolizing enzymes. Cytotoxicity may also play a role, particularly in affecting induction of xenobiotic-metabolizing enzymes. The effects of interactions manifested at the level of the expression of toxic endpoints may result from interactions at the metabolic level or may have other causes. New approaches to genome-wide effects (e.g., microarray studies) are also discussed.

Toxins and venoms.

Toxins are produced by numerous microorganisms and invertebrates as well as by higher plants and animals. Venoms are produced by many groups of animals, from coelenterates to vertebrates. While toxins and venoms are the primary toxicological concern in natural ecosystems, they are frequently of importance in agroecosystems and in military deployments. They belong to a very large number of chemical classes and consequently are usually classified according to the groups of organisms producing them, for example, mycotoxins, algal toxins, and insect venoms. Plant toxins are representative of a larger group of phytochemicals known as secondary plant chemicals or plant allelochemicals. They may be acutely toxic, have chronic toxicity, or may be toxic at one dose but therapeutic at a lower dose. Some drugs of abuse are plant toxins abuse. A representative selection of the more important members of each category is provided.

Summary and conclusions.

The complexity and interrelationships of human environments are constantly undergoing further definition. The importance of such definition can be realized when such broad environmental categories as natural ecosystems, the agroecosystem, occupational, urban, domestic, and military deployments, all of which can be subdivided into more specialized environments, are considered. Set against this background, it is clear that the principal role of toxicology is risk analysis. Risk analysis consists not only of risk assessment but also risk management and risk communication. New paradigms in hazard assessment are revolutionizing risk assessment, and all aspects, from molecular toxicology to outreach programs, will be involved in future considerations of human environments.

Human variation in CYP-specific chlorpyrifos metabolism.

Chlorpyrifos, an organophophorothioate insecticide, is bioactivated to the neurotoxic metabolite, chlorpyrifos-oxon (CPO) by cytochromes P450 (CYPs). To determine the variability in chlorpyrifos bioactivation, CPO production by human liver microsomes from 17 individual donors was compared relative to phenotype and genotype. CPO production varied over 14-fold between individuals in incubations utilizing 20 µM chlorpyrifos as substrate, while CPO production varied 57-fold in incubations with 100 µM chlorpyrifos. For all but two samples, the formation of the less toxic metabolite, 3,5,6-trichloro-2-pyridinol (TCP), was greater than CPO production. TCP production varied 9-fold in incubations utilizing 20 µM chlorpyrifos as substrate and 19-fold using 100 µM chlorpyrifos. Chlorpyrifos metabolism by individual human liver microsomes was significantly correlated with CYP2B6, CYP2C19 and CYP3A4 related activity. CPO formation was best correlated with CYP2B6 related activity at low (20 µM) chlorpyrifos concentrations while CYP3A4 related activity was best correlated with CPO formation at high concentrations (100 µM) of chlorpyrifos. TCP production was best correlated with CYP3A4 activity at all substrate concentrations of chlorpyrifos. The production of both CPO and TCP was significantly lower at a concentration of 20 µM chlorpyrifos as compared to 100 µM chlorpyrifos. Calculations of percent total normalized rates (% TNR) and the chemical inhibitors ketoconazole and ticlopidine were used to confirm the importance of CYP2B6, CYP2C19, and CYP3A4 for the metabolism of chlorpyrifos. The combination of ketoconazole and ticlopidine inhibited the majority of TCP and CPO formation. CPO formation did not differ by CYP2B6 genotype. Individual variations in CPO production may need to be considered in determining the risk of chlorpyrifos poisoning.

Human hepatic CYP2B6 developmental expression: the impact of age and genotype.

Although CYP2B6 is known to metabolize numerous pharmaceuticals and toxicants in adults, little is known regarding CYP2B6 ontogeny or its possible role in pediatric drug/toxicant metabolism. To address this knowledge gap, hepatic CYP2B6 protein levels were characterized in microsomal protein preparations isolated from a pediatric liver bank (N=217). Donor ages ranged from 10 weeks gestation to 17 years of age with a median age of 1.9 months. CYP2B6 levels were measured by semi-quantitative western blotting. Overall, CYP2B6 expression was detected in 75% of samples. However, the percentage of samples with detectable CYP2B6 protein increased with age from 64% in fetal samples to 95% in samples from donors >10 years of age. There was a significant, but only 2-fold increase in median CYP2B6 expression after the neonatal period (birth to 30 days postnatal) although protein levels varied over 25-fold in both age groups. The median CYP2B6 level in samples over 30 postnatal days to 17 years of age (1.3 pmol/mg microsomal protein) was lower than previously reported adult levels (2.2-22 pmol/mg microsomal protein), however, this likely relates to the median age of these samples, i.e., 10.3 months. CYP2B6 expression did not vary significantly by gender. Furthermore, CYP2B6 levels did not correlate with CYP3A4, CYP3A5.1 or CYP3A7 activity, consistent with different mechanisms controlling the ontogeny and constitutive expression of these enzymes and the lack of significant induction in the pediatric samples.

Metabolic interactions of agrochemicals in humans.

Agrochemicals and other xenobiotics are metabolized by xenobiotic-metabolizing enzymes (XMEs) to products that may be more or less toxic than the parent chemical. In this regard, phase-I XMEs such as cytochrome P450s (CYPs) are of primary importance. Interactions at the level of metabolism may take place via either inhibition or induction of XMEs. Such interactions have often been investigated, in vitro, in experimental animals, using subcellular fractions such as liver microsomes, but seldom in humans or at the level of individual XME isoforms. The authors have been investigating the metabolism of a number of agrochemicals by human liver microsomes and recombinant CYP isoforms and have recently embarked on studies of the induction of XMEs in human hepatocytes. The insecticides chlorpyrifos, carbaryl, carbofuran and fipronil, as well as the repellant DEET, are all extensively metabolized by human liver microsomes and, although a number of CYP isoforms may be involved, CYP2B6 and CYP3A4 are usually the most important. Permethrin is hydrolyzed by esterase(s) present in both human liver microsomes and cytosol. A number of metabolic interactions have been observed. Chlorpyrifos and other phosphorothioates are potent inhibitors of the CYP-dependent metabolism of both endogenous substrates, such as testosterone and estradiol, and exogenous substrates, such as carbaryl, presumably as a result of the interaction of highly reactive sulfur, released during the oxidative desulfuration reaction, with the heme iron of CYP. The hydrolysis of permethrin in human liver can be inhibited by chlorpyrifos oxon and by carbaryl. Fipronil can inhibit testosterone metabolism by CYP3A4 and is an effective inducer of CYP isoforms in human hepatocytes.

Enzyme induction and cytotoxicity in human hepatocytes by chlorpyrifos and N,N-diethyl-m-toluamide (DEET).

Xenobiotics, including drugs and environmental chemicals, can influence cytochrome P450 (CYP) levels by altering the transcription of CYP genes. To minimize potential drug-pesticide and pesticide-pesticide interactions it is important to evaluate the potential of pesticides to induce CYP isoforms and to cause cytotoxicity in humans. The present study was designed to examine chlorpyrifos and DEET mediated induction of CYP isoforms and also to characterize their potential cytotoxic effects on primary human hepatocytes. DEET significantly induced CYP3A4, CYP2B6, CYP2A6 and CYP1A2 mRNA expression while chlorpyrifos induced CYP1A1, CYP1A2 and CYP3A4 mRNA, and to a lesser extent, CYP1B1 and CYP2B6 mRNA in primary human hepatocytes. Chlorpyrifos and DEET also mediated the expression of CYP isoforms, particularly CYP3A4, CYP2B6 and CYP1A1, as shown by CYP3A4-specific protein expression, testosterone metabolism and CYP1Al-specific activity assays. DEET is a mild, while chlorpyrifos is a relatively potent, inducer of adenylate kinase and caspase-3/7, an indicator of apoptosis, while inducing 15-20% and 25-30% cell death, respectively. Therefore, DEET and chlorpyrifos mediated induction of CYP mRNA and functional CYP isoforms together with their cytotoxic potential in human hepatocytes suggests that exposure to chlorpyrifos and/or DEET should be considered in human health impact analysis.

Pyrethroids: cytotoxicity and induction of CYP isoforms in human hepatocytes.

Deltamethrin [(S)-alpha-cyano-3-phenoxybenzyl-cis-(1 R,3R)-3(2,2-dibromovinyl)(2,2-dimethyl-cyclopropane-carboxylate] and permethrin [3-phenoxybenzyl(1RS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropanecarboxylate] are pyrethroid insecticides used in agriculture, public health and military deployments. Pyrethroids are known to be capable of inducing cytochrome P450 (CYP) 2B1/2B2, CYP1A1 and overall CYP content in rat liver. The objectives of this study were to evaluate the potential of deltamethrin and permethrin to cause cytotoxicity and to induce CYP isoforms in human hepatocytes. Permethrin and deltamethrin showed dose-dependent effects on adenylate kinase activity in HepG2 cells, in which 50 and 100 microM doses, respectively, induced a 3-5 fold increase in activity, and also induced adenylate kinase activity in primary human hepatocytes. An approximately 3-fold induction was noted at 200 microM deltamethrin and a 4-fold induction at 100 microM permethrin. Cytotoxicity was noted in HepG2 cells following 48-72 h exposure to 100 or 200 microM deltamethrin and permethrin, respectively. Dose-dependent induction of caspase-3/7 was initiated by 12.5 microM deltamethrin or by 3.125 microM permethrin. Actinomycin D, a positive control for induction of caspase 3/7, induced caspase-3/7, an effect completely abrogated by the specific inhibitor Z-DEVD-FMK. At 100 microM deltamethrin 2-3 fold induction of CYP1A1 and CYP2B6 mRNA was observed, while at the same time an approximately 25-fold induction of CYP3A4 was noted. Permethrin-mediated CYP induction was much less potent, 4-fold or less for CYP1A1, CYP3A4, CYP3A5, CYP2B6 and CYP2A6. It has also been shown that these pyrethroids are ligands for the pregnane X receptor (PXR).

Human metabolic interactions of environmental chemicals.

Investigations utilizing recombinant human xenobiotic-metabolizing enzymes as well as human hepatocytes have revealed a number of interactions not only between different environmental chemicals (ECs) but also between ECs and endogenous metabolites. Organophosphorus insecticides (OPs) are potent inhibitors of the human metabolism of carbaryl, carbofuran, DEET and fipronil, as well as the jet fuel components, nonane and naphthalene. OPs are potent irreversible inhibitors of testosterone metabolism by cytochrome P450 (CYP) 3A4 and of estradiol metabolism by CYP3A4 and CYP1A2. All of these CYP inhibitions are believed to be due to the release of reactive sulfur during CYP-catalyzed oxidative desulfuration. It has also been shown that the esterase(s) responsible for the initial step in permethrin metabolism in human liver is inhibited by both chlorpyrifos oxon and carbaryl. A number of pesticides, including chlorpyrifos, fipronil and permethrin, and the repellent, DEET, have been shown to be inducers of CYP isoforms in human hepatocytes, with fipronil being the most potent. Several agrochemicals, including fipronil and the pyrethroids, permethrin and deltamethrin, show toxicity toward human hepatocytes with fipronil being the most potent in this regard. Endosulfan-alpha, which has shown promise as a model substrate for phenotyping CYP3A4 and CYP2B6 in human liver microsomes, is also an inducer of CYP2B6, acting through the PXR receptor.

Inhibition of fipronil and nonane metabolism in human liver microsomes and human cytochrome P450 isoforms by chlorpyrifos.

Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based.

The importance of cytochrome P450 2B6 in the human metabolism of environmental chemicals.

Cytochrome P450 (CYP) 2B6 (CYP2B6) is a human CYP isoform found in variable amounts in the liver and other organs. It is known to be inducible and polymorphic and has a wide range of xenobiotic substrates. Studies of CYP2B6 to date have concentrated heavily on clinical drugs. In the present communication, however, we concentrate on its role in the metabolism of environmental xenobiotics. The term environment is used, in its broadest sense, to include natural ecosystems and agroecosystems as well as the industrial and indoor domestic environments. In essence, this excludes only clinical drugs and drugs of abuse. Many of these chemicals, including agrochemicals and industrial chemicals, can serve as substrates, inhibitors and/or inducers of CYP2B6, these activities being often modified by the existence of polymorphic variants. Metabolism-based interactions between environmental chemicals are discussed, as well as the emerging possibility of metabolic interactions between environmental chemicals and clinical drugs.

The effect of chlorpyrifos-oxon and other xenobiotics on the human cytochrome P450-dependent metabolism of naphthalene and deet.

Chlorpyrifos-oxon (CPO), a metabolite of chlorpyrifos, is a potent inhibitor of acetylcholinesterase and, although the neurotoxicological impact of this organophosphorus compound has been broadly studied both in vitro and in vivo, there are few studies of metabolic interactions of CPO with other xenobiotics. CPO significantly activated the production of 1-naphthol (5-fold), 2-naphthol (10-fold), trans-1,2-dihydro-1,2-naphthalenediol (1.5-fold), and 1,4-naphthoquinone from naphthalene by human liver microsomes (HLM). It was further demonstrated that the production of naphthalene metabolites by CYP2C8, 2C9*(1), 2C19, 2D6*(1), 3A4, 3A5, and 3A7 was activated by CPO, while the production of naphthalene metabolites by CYP1A1, 1A2, 1B1, and 2B6 was inhibited by CPO. CPO inhibited CYP1A2 production of naphthalene metabolites, while activating their production by CYP3A4. Similarly, CPO inhibited the production of N,N-diethyl-m-hydroxymethylbenzamide (BALC) from DEET by human liver microsomes, but activated the production of N-ethyl-m-toluamide (ET) from this substrate. CYP2B6, the most efficient isoform for BALC production, was inhibited by CPO, while CYP3A4, the most efficient isoform for ET production, was activated by CPO. CPO inhibited CYP2B6 production of both BALC and ET from DEET, but activated CYP3A4 production of ET, while inhibiting CYP3A4 BALC production. CPO appears to facilitate the binding of naphthalene to CYP3A4. This metabolic activation is independent of cytochrome b5, suggesting that activation of CYP3A4 by CPO is associated with a conformational change of the isoform rather than facilitating electron transfer.

Metabolism of chlorpyrifos and chlorpyrifos oxon by human hepatocytes.

The metabolism of chlorpyrifos (CPS) and chlorpyrifos oxon (CPO) by human hepatocytes and human liver S9 fractions was investigated using LC-MS/MS. Cytochrome P450 (CYP)-dependent and phase II-related products were determined following incubation with CPS and CPO. CYP-related products, 3,5,6-trichloro-2-pyridinol (TCP), diethyl thiophosphate, and dealkylated CPS, were found following CPS treatment and dealkylated CPO following CPO treatment. Diethyl phosphate was not identified because of its high polarity and lack of retention with the chromatographic conditions employed. Phase II-related conjugates, including O- and S-glucuronides as well as 11 GSH-derived metabolites, were identified in CPS-treated human hepatocytes, although the O-sulfate of TCP conjugate was found only when human liver S9 fractions were used as the enzyme source. O-Glucuronide of TCP was also identified in CPO-treated hepatocytes. CPS and CPO were identified using HPLC-UV after CPS metabolism by the human liver S9 fraction. However, CPO was not found following treatment of human hepatocytes with either CPS or CPO. These results suggest that human liver plays an important role in detoxification, rather than activation, of CPS.