Role of Noncoding RNAs in Acetaminophen-Induced Liver Injury.

Genomic and transcriptomic analyses have well established that the major fraction of the mammalian genome is transcribed into different classes of RNAs ranging in size from a few nucleotides to hundreds of thousands of nucleotides, which do not encode any protein. Some of these noncoding RNAs (ncRNAs) are directly or indirectly linked to the regulation of expression or functions of 25,000 proteins coded by <2% of the human genome. Among these regulatory RNAs, microRNAs are small (2125 nucleotides) RNAs that are processed from precursor RNAs that have stemloop structure, whereas noncoding RNAs >200 nucleotides are termed long noncoding RNAs (lncRNAs). Circular RNAs (circRNAs) are newly identified lncRNA members that are generated by back-splicing of primary transcripts. The functions of ncRNAs in modulating liver toxicity of xenobiotics are emerging only recently. Acetaminophen (N-acetyl-para-aminophenol, paracetamol or APAP) is a safe analgesic and antipyretic drug at the therapeutic dose. However, it can cause severe liver toxicity that may lead to liver failure if overdosed or combined with alcohol, herbs, or other xenobiotics. This review discusses the role of ncRNAs in acetaminophen metabolism, toxicity, and liver regeneration after APAP-induced liver injury (AILI).

Transcriptome analysis of antennal cytochrome P450s and their transcriptional responses to plant and locust volatiles in Locusta migratoria.

Cytochrome P450 monooxygenases (P450s) constitute a large superfamily of heme-thiolate proteins that are involved in the biosynthesis or degradation of endogenous compounds and detoxification of exogenous chemicals. It has been reported that P450s could serve as odorant-degrading enzymes (ODEs) to inactivate odorants to avoid saturating the antennae. However, there is little information about P450s in the antennae of Locusta migratoria. In the current work, we conducted an antenna transcriptome analysis and identified 92 P450s, including 68 full-length and 24 partial sequences. Phylogenetic analysis showed that 68 full-length P450s were grouped into four clans: CYP2, CYP3, CYP4, and mitochondria clans. Tissue, stage, and sex-dependent expressions of these 68 P450s were investigated. The results showed that 4 P450s were antenna-specific, whereas others were antenna-rich but also expressed in other tissues, implying their various potential roles in the antennae. In addition, the responses of seven selected P450s to five gramineous plant volatiles and four locust volatiles were determined. CYP6MU1 could be induced by almost all compounds tested, suggesting its important roles in odorant processing. Different P450s exhibited diverse responses to odorants, indicating that specific regulation of P450 expression by odorants might modulate the sensitivity of the olfactory responses to various chemicals.

Hepatic PGC-1α is not essential for fasting-induced cytochrome p450 regulation in mouse liver.

Fasting has been shown to regulate the expression of the cytochrome p450 (CYP) enzyme system in the liver. However, the exact mechanism behind the fasting-induced regulation of the CYP's remains unknown. In the present study we tested the hypothesis that the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), which is a key-regulator of energy metabolism, is responsible for the fasting-induced regulation of the CYP's. Lox/lox and liver specific PGC-1α (LKO) mice of both sexes, fasted for 18 h and the content of the CYP's as well as the hepatic metabolome was assessed. Fasting increased the mRNA content of Cyp2a4, Cyp2e1, Cyp3a11 and Cyp4a10. The fasting-induced response in Cyp4a10 mRNA content was different between lox/lox and LKO mice, while the absence of PGC-1α had no effect on the fasting-induced response for the other Cyp's. Moreover, the fasting-induced response in mRNA content of Sirtinus 1 and Perilipin 2 was different between lox/lox and LKO mice. Only the CYP1A isoform showed a fasting-induced response at the protein level. Absence of hepatic PGC-1α had no effect on the apparent metabolome, where fasting vs fed was the only discriminate in the following multivariate analysis. In conclusion, hepatic PGC-1α is not essential for the fasting-induced regulation of hepatic CYP's.

Effects of anemoside B4 on pharmacokinetics of florfenicol and mRNA expression of CXR, MDR1, CYP3A37 and UGT1E in broilers.

Pulsatillae radix, a traditional Chinese medicine (TCM), is often used in combination with florfenicol for treatment of intestinal infection in Chinese veterinary clinics. Anemoside B4 (AB4) is the major effective saponin in Pulsatillae radix. This study aimed to investigate whether the pharmacokinetics of florfenicol in broilers was affected by the combination of AB4. In this study, broilers were given AB4 (50 mg/kg BW), or 0.9% sodium chloride solution by oral administration for 7 days. They were then fed florfenicol orally (30 mg/kg BW) on the eighth day. The results showed that the AUC(0-∞), MRT(0-∞), t1/2z and Cmax of florfenicol were significantly decreased, and the Vz/F and CLz/F were significantly increased by AB4; the mRNA expression levels of CXR, CYP3A37 and MDR1 (except CXR and CYP3A37 in the liver) were up-regulated by AB4. In conclusion, AB4 altered the pharmacokinetics of florfenicol, resulting in lower plasma concentrations of florfenicol, this was probably related to the mRNA expression of CXR, CYP3A37 and MDR1 in the jejunum and liver (except CXR and CYP3A37) increased by AB4. The implications of these findings on the effect of traditional Chinese medicine containing AB4 on the effectiveness of florfenicol in veterinary practice deserve study.

Effect of piperine and quercetin alone or in combination with marbofloxacin on CYP3A37 and MDR1 mRNA expression levels in broiler chickens.

After oral route of administration, drug absorption is unpredictable and is governed by various factors such as multi drug resistance-1 (MDR1) an efflux transporter and drug metabolizing enzymes (like CYP3A4, CYP3A37, CYP2D6) at intestine and liver. Naturally available phyto chemicals like piperine and quercetin as well as some floroquinolones are known to inhibit MDR1 and CYP3A37 activity and increases bioavailability of co-administered drugs. This study was carried out to investigate the effect of piperine and quercetin alone or in combination with marbofloxacin on CYP3A37 and MDR1 mRNA expression levels in liver and intestine of broiler chicken. After oral administration of piperine and quercetin for 3 consecutive days followed by with or without oral administration of marbofloxacin for 5 days, CYP3A37 and MDR1 mRNA expression levels were determined using quantitative real-time PCR. Total of 36 broiler chickens in seven individual groups were treated with different regimen and the mRNA expression levels at duodenum and liver were analyzed with apt statistical tools. After piperine and quercetin combined treatment with marbofloxacin, CYP3A37 mRNA expression levels were significantly down regulated by 20.57 (p = .034) and 25.95 (p = .003) folds; and MDR1 mRNA expression levels were also significantly down regulated by 11.33 (p = .012) and 33.59 (p = .006) folds in liver and duodenum, respectively. Down regulation of CYP3A37 and MDR1 mRNA in liver and duodenum indicate the combined pretreatment of piperine and quercetin may be useful for improving the pharmacokinetics of orally administered drugs which are substrates for CYP3A37 and MDR1.

Tissue distribution and characterization of feline cytochrome P450 genes related to polychlorinated biphenyl exposure.

Cats have been known to be extremely sensitive to chemical exposures. To understand these model species' sensitivity to chemicals and their toxicities, the expression profiles of xenobiotic-metabolizing enzymes should be studied. Unfortunately, the characterization of cytochrome P450 (CYP), the dominant enzyme in phase I metabolism, in cats has not extensively been studied. Polychlorinated biphenyls (PCBs) are known as CYP inducers in animals, but the information regarding the PCB-induced CYP expression in cats is limited. Therefore, in the present study, we aimed to elucidate the mRNA expression of the CYP1-CYP3 families in the cat tissues and to investigate the CYP mRNA expression related to PCB exposure. In cats, the greatest abundance of CYP1-CYP3 (CYP1A2, CYP2A13, CYP2C41, CYP2D6, CYP2E1, CYP2E2, CYP2F2, CYP2F5, CYP2J2, CYP2U1, and CYP3A132) was expressed in the liver, but some extrahepatic isozymes were found in the kidney (CYP1A1), heart (CYP1B1), lung (CYP2B11 and CYP2S1) and small intestine (CYP3A131). In cats, CYP1A1, CYP1A2 and CYP1B1 were significantly upregulated in the liver as well as in several tissues exposed to PCBs, indicating that these CYPs were distinctly induced by PCBs. The strong correlations between 3,3',4,4'-tetrachlorobiphenyl (CB77) and CYP1A1 and CYP1B1 mRNA expressions were noted, demonstrating that CB77 could be a potent CYP1 inducer. In addition, these CYP isoforms could play an essential role in the PCBs biotransformation, particularly 3-4 Cl-PCBs, because a high hydroxylated metabolite level of 3-4 Cl-OH-PCBs was observed in the liver.

Effects of lactic acid on drug-metabolizing enzymes in Chinese mitten crab (Eriocheir sisnensis) after oral enrofloxacin.

Enrofloxacin (ENR) is the most commonly used antibiotic in crustacean farming in China. Diet supplementation with lactic acid (LA) may, however, affect the efficacy and safety of ENR-based drugs. The aims of this study were to investigate the effects of LA on drug residues and elimination of oral ENR in Chinese mitten crab (Eriocheir sinensis) and to determine ENR and gene expression levels of drug-metabolizing enzymes in the hepatopancreas. To this end, ENR was orally administered to the crabs at a dose of 10.0 mg kg-1 body weight on the eighth day after feeding diets supplemented with 0.3%LA. The results showed that ENR levels in the hepatopancreas were significantly different at 1 and 12 h between the ENR and ENR + 0.3% LA groups (P < 0.05). Lactic acid did not significantly affect the expression of CYP2A (phase I). However, the expressions of CYP3 (phase I) and GST (phase II) were significantly up-regulated by LA during the elimination process of ENR (6-24 h). At Tmax (1 h), the expression of phosphoenolpyruvate carboxykinase (PEPCK) was induced and expression of succinate dehydrogenase (SDH) was inhibited by LA. Both of these enzymes were significantly inhibited during the elimination process of ENR. The results suggest that LA contributes to the elimination of ENR, and thus, enhances hepatopancreas biotransformation and anti-injury capacity in E. sinensis.

Investigation of the inhibition of eight major human cytochrome P450 isozymes by a probe substrate cocktail in vitro with emphasis on CYP2E1.

1. A protocol has been developed and validated for the high-throughput screening of eight major human cytochrome P450 (CYP) isozymes inhibition (CYP 1A2, 2C9, 2C19, 2D6, 3A4, 2B6, 2C8 and 2E1) using an in vitro probe cocktail containing eight substrates by overcoming the unfavorable effect of assay conditions on CYP2E1 inhibition data. 2. The cocktail consisting of selective probe substrates like tacrine (CYP1A2), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A4), bupropion (CYP2B6), paclitaxel (CYP2C8) and chlorzoxazone (CYP2E1) was incubated with human liver microsomes. 3. The method was investigated by incubating well-known CYP inhibitors {alphanaphthoflavone (CYP1A2), sulfaphenazole (CYP2C9), N-3-benzylnirvanol (CYP2C19), quinidine (CYP2D6), ketoconazole (CYP3A4), ticlopidine (CYP2B6), quercetin (CYP2C8) and 4-methylpyrazole (CYP2E1)} with the substrate cocktail. A fast gradient liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for this study. 4. The IC50 values determined for typical CYP inhibitors were reproducible and consistent with those in the literature. DMSO has significant effect and itself inhibits CYP2E1. DMSO should not exceed 0.1% for the determination of reliable CYP2E1 inhibition profile. This cocktail assay offers an efficient and robust method to determine the CYP450 isoforms inhibition profiles of large numbers of compounds in a quick turnaround time.

Differences in diet and biotransformation enzymes of coral reef butterflyfishes between Australia and Hawaii.

Many reef fishes are capable of feeding on chemically-defended benthic prey, such as soft (alcyonarian) corals; however, little is known about the molecular mechanisms that underpin allelochemical biotransformation and detoxification. Butterflyfishes (Chaetodon: Chaetdontidae) are a useful group for comparatively exploring links between biotransformation enzymes and diet, because they commonly feed on chemically defended prey. Moreover, diets of some species vary among geographic locations. This study compares gene expression, protein and enzymatic activity of key detoxification enzymes (cytochrome P450 (CYP) 2, 3, epoxide hydrolase, glutathione transferase and UDP-glucuronosyltransferase) in livers of four coral-feeding butterflyfish species between Australia and Hawaii, where these fishes differ in diet composition. For C. kleinii, C. auriga, and C. unimaculatus, we found higher CYP2 and CYP3 levels were linked to more allelochemically rich diets in Australia relative to Hawaii. For C. lunulatus from Hawaii CYP2 and CYP3 levels were 1 to 20-fold higher than C. lunulatus from Australia, possibly due to their predominant prey in Hawaii (Porities spp.) being richer in allelochemicals. UGT, GST and epoxide hydrolase varied between species and location and did not correspond to any specific dietary preference or location. Higher levels of CYP2 and CYP3A isozymes in species that feed on allelochemically-rich prey suggest that these biotransformation enzymes may be involved in detoxification of coral dietary allelochemicals in butterflyfishes.

Polydatin alleviated alcoholic liver injury in zebrafish larvae through ameliorating lipid metabolism and oxidative stress.

Hepatic steatosis is the early stage of alcoholic liver disease (ALD), may progress to steatohepatitis, fibrosis even cirrhosis. Polydatin, the primary active component of Polygonum cuspidatum Sieb. et Zucc, has been recognized to possess hepatoprotective and anti-inflammatory properties. To investigate whether polydatin alleviates ethanol induced liver injury and to elucidate the underlying molecular mechanisms, zebrafish larvae at 4 days post-fertilization (dpf) were exposed to 350 mmol/L of ethanol for 32 h, then treated with polydatin for 48 h. Oil red O, Nile Red and H&E staining were used to analyze the pathological changes in liver. The mRNA levels were measured by quantitative PCR and the antioxidant capacity was detected using H2O2-specific fluorescent probe. Here, polydatin strongly alleviated hepatic steatosis and decreased the expression levels of alcohol and lipid metabolism-related genes, including CYP2Y3, CYP3A65, HMGCRa, HMGCRb and FASN. Additionally, polydatin inhibited oxidative stress in the liver according to fluorescent probe. Moreover, significantly up-regulated expression of DNA damage-related genes (CHOP, GADD45αa) revealed that polydatin attenuated hepatic apoptosis in larvae. In conclusion, polydatin may improve the liver function of zebrafish with acute alcoholic liver injury through attenuating hepatic fat accumulation, ameliorating lipid and ethanol metabolism and reducing oxidative stress and DNA damage.

The metabolism distribution and effect of imidacloprid in chinese lizards (Eremias argus) following oral exposure.

Systematically evaluation of the metabolism, distribution and effect of imidacloprid in Chinese lizards (Eremias argus) were carried out following oral exposure. Imidacloprid-olefin-guanidine was prone to accumulate in the brain and caused potential neurotoxicity. Percutaneous and excretory excretions were the primary ways for the elimination of imidacloprid and its metabolites. Liver was the main site for hydroxy reduction and nitro-reduction metabolism of imidacloprid. The metabolism of imidacloprid was a complex process in which many metabolic enzymes participated. Aldehyde oxidase and CYP2C9 were the key enzymes in nitro-reduction process. CYP3A4 dominated the process of hydroxylation and desaturation. The increase in Glutathione S-transferase expression may be related to the removal of imidacloprid, but also related to the oxidative stress reaction that imidacloprid may cause in tissues, especially in the kidney. The findings enrich and supplement the knowledge of the environmental fate of imidacloprid in reptiles.

High-throughput metabolism-induced toxicity assays demonstrated on a 384-pillar plate.

The US Environmental Protection Agency (EPA) launched the Transform Tox Testing Challenge in 2016 with the goal of developing practical methods that can be integrated into conventional high-throughput screening (HTS) assays to better predict the toxicity of parent compounds and their metabolites in vivo. In response to this need and to retrofit existing HTS assays for assessing metabolism-induced toxicity of compounds, we have developed a 384-pillar plate that is complementary to traditional 384-well plates and ideally suited for culturing human cells in three dimensions at a microscale. Briefly, human embryonic kidney (HEK) 293 cells in a mixture of alginate and Matrigel were printed on the 384-pillar plates using a microarray spotter, which were coupled with 384-well plates containing nine model compounds provided by the EPA, five representative Phase I and II drug metabolizing enzymes (DMEs), and one no enzyme control. Viability and membrane integrity of HEK 293 cells were measured with the calcein AM and CellTiter-Glo® kit to determine the IC50 values of the nine parent compounds and DME-generated metabolites. The Z' factors and the coefficient of variation measured were above 0.6 and below 14%, respectively, indicating that the assays established on the 384-pillar plate are robust and reproducible. Out of nine compounds tested, six compounds showed augmented toxicity with DMEs and one compound showed detoxification with a Phase II DME. This result indicates that the 384-pillar plate platform can be used to measure metabolism-induced toxicity of compounds in high-throughput with individual DMEs. As xenobiotics metabolism is a complex process with a variety of DMEs involved, the predictivity of our approach could be further improved with mixtures of DMEs.

Prescription Opioid Fatalities: Examining Why the Healer Could be the Culprit.

Prescription opioid use has increased rapidly in developed countries, as have fatalities and other related adverse events. This review examines the intrinsic characteristics of opioids, including their mechanisms of action and pharmacokinetic and pharmacodynamic properties, to determine how the use of a regonised pharmacological remedy for a medically confirmed ailment could result in an accidental fatality. Opioids trigger biological processes that inhibit their own therapeutic effect. Prolonged use of opioids can result in activation of pronociceptive systems, leading to opioid-induced hyperalgesia and tolerance, while opioid metabolites can antagonise the antinociceptive action of the parent drug, also leading to opioid-induced hyperalgesia and tolerance. Pain stimulates respiration and counteracts the respiratory depression effect of opioids. Analgesia from opioids leads to loss of this protective mechanism, leading to increased risk of death due to respiratory failure. Increased patient counseling during opioid prescribing and dispensing, and limiting prescription to short-term use in non-malignant pain, may decrease the adverse effects of opioids. The vast majority of patients who unintentionally experience serious adverse events from pharmaceutical opioids do not start out as drug seekers. Even opioid use within prescribing guidelines can place some patients at risk of death and may prevent patients from seeking help for prescription opioid dependence.

Emerging roles for brain drug-metabolizing cytochrome P450 enzymes in neuropsychiatric conditions and responses to drugs.

P450s in the human brain were originally considered unlikely to contribute significantly to the clearance of drugs and other xenobiotic chemicals, since their overall expression was a small fraction of that found in the liver. However, it is now recognized that P450s play substantial roles in the metabolism of both exogenous and endogenous chemicals in the brain, but in a highly cell type- and region-specific manner, in line with the greater functional heterogeneity of the brain compared to the liver. Studies of brain P450 expression and the characterization of the catalytic activity of specific forms expressed as recombinant enzymes have suggested possible roles for xenobiotic-metabolizing P450s in the brain. It is now possible to confirm these roles through the use of intracerebroventricular administration of selective P450 inhibitors in animal models, coupled with brain sampling techniques to measure drug concentrations in vivo, and modern neuroimaging techniques. The purpose of this review is to discuss the evidence behind the functional importance of P450s from the "xenobiotic-metabolizing" families, CYP1, CYP2 and CYP3 in the brain. Approaches used to define the quantitative and qualitative significance of these P450s in determining tissue-specific levels of xenobiotics in brain will be considered. Finally, the possible roles of these enzymes in brain biochemistry will be examined in light of the demonstrated activity of these enzymes in vitro and the association of particular P450 forms with disease states.

Potential pharmacokinetic effect of rifampicin on enrofloxacin in broilers: Roles of P-glycoprotein and BCRP induction by rifampicin.

P-glycoprotein ( P-GP: , encoding gene Abcb1) and Breast Cancer Resistance Protein ( BCRP: , encoding gene Abcg2) are transport proteins that play a major role in modulating the bioavailability of oral drugs in humans and rodents. It has been shown that rifampicin is the typical inducer of P-gp in rodents by activating the nuclear receptor. However, its effect on Abcb1, Abcg2, CYP3A, and chicken xenobiotic-sensing orphan nuclear receptor ( CXR: ) mRNA expression in broilers is poorly understood. This study explored the effect of rifampicin on mRNA expression of Abcb1, Abcg2, CYP3A37, CXR as well as its effect on the pharmacokinetics of enrofloxacin in broilers. The mRNA levels of Abcb1, Abcg2, CYP3A37, and CXR were significantly increased in the liver (except Abcg2), kidney, jejunum, and ileum (P < 0.05) but not significantly changed in the duodenum (P > 0.05) after treated with rifampicin. Further analysis revealed that the variation tendencies of Abcb1, Abcg2, and CYP3A37 expression levels were significantly correlated with CXR mRNA expression levels in liver, kidney, jejunum, and ileum. Coadministration of rifampicin significantly changed the pharmacokinetic behavior of enrofloxacin orally administered by showing clearly lower AUC0-∞, AUC0-t, and Cmax as well as longer Tmax. The bioavailability of orally administered enrofloxacin was decreased from 72.5% to 24.8% by rifampicin. However, rifampicin did not significantly change the pharmacokinetics of enrofloxacin following intravenous administration. Our study shows that rifampicin up-regulated the small intestinal level of P-gp and BCRP and suggests that P-gp and BCRP are key factors that affected pharmacokinetic behavior of orally administered enrofloxacin by limiting its absorption from the intestine in broilers.