Increased Cytochrome P450 Activity Research Articles

RGD density along with substrate stiffness regulate hPSC hepatocyte functionality through YAP signalling

Human pluripotent stem cell-derived hepatocytes (hPSC-Heps) may be suitable for treating liver diseases, but differentiation protocols often fail to yield adult-like cells. We hypothesised that replicating healthy liver niche biochemical and biophysical cues would produce hepatocytes with desired metabolic functionality. Using 2D synthetic hydrogels which independently control mechanical properties and biochemical cues, we found that culturing hPSC-Heps on surfaces matching the stiffness of fibrotic liver tissue upregulated expression of genes for RGD-binding integrins, and increased expression of YAP/TAZ and their transcriptional targets. Alternatively, culture on soft, healthy liver-like substrates drove increases in cytochrome p450 activity and ureagenesis. Knockdown of ITGB1 or reducing RGD-motif-containing peptide concentration in stiff hydrogels reduced YAP activity and improved metabolic functionality; however, on soft substrates, reducing RGD concentration had the opposite effect. Furthermore, targeting YAP activity with verteporfin or forskolin increased cytochrome p450 activity, with forskolin dramatically enhancing urea synthesis. hPSC-Heps could also be successfully encapsulated within RGD peptide-containing hydrogels without negatively impacting hepatic functionality, and compared to 2D cultures, 3D cultured hPSC-Heps secreted significantly less fetal liver-associated alpha-fetoprotein, suggesting furthered differentiation. Our platform overcomes technical hurdles in replicating the liver niche, and allowed us to identify a role for YAP/TAZ-mediated mechanosensing in hPSC-Hep differentiation.

Validation of the Anticolitis Efficacy of the Jian-Wei-Yu-Yang Formula.

Background Inflammatory bowel disease (IBD) is a major cause of morbidity and mortality due to its repetitive remission and relapse. The Jian-Wei-Yu-Yang (JW) formula has a historical application in the clinic to combat gastrointestinal disorders. The investigation aimed to explore the molecular and cellular mechanisms of JW. Methods 2% dextran sodium sulfate (DSS) was diluted in drinking water and given to mice for 5 days to establish murine models of experimental colitis, and different doses of JW solution were administered for 14 days. Network pharmacology analysis and weighted gene co-expression network analysis (WGCNA) were utilized to predict the therapeutic role of JW against experimental colitis and colitis-associated colorectal cancer (CAC). 16S rRNA sequencing and untargeted metabolomics were conducted using murine feces. Western blotting, immunocytochemistry, and wound healing experiments were performed to confirm the molecular mechanisms. Results (1) Liquid chromatography with mass spectrometry was utilized to confirm the validity of the JW formula. The high dose of JW treatment markedly attenuated DSS-induced experimental colitis progression, and the targets were enriched in inflammation, infection, and tumorigenesis. (2) The JW targets were related to the survival probability in patients with colorectal cancer, underlying a potential therapeutic value in CRC intervention. (3) Moreover, the JW therapy successfully rescued the decreased richness and diversity of microbiota, suppressed the potentially pathogenic phenotype of the gut microorganisms, and increased cytochrome P450 activity in murine colitis models. (4) Our in vitro experiments confirmed that the JW treatment suppressed caspase3-dependent pyroptosis, hypoxia-inducible factor 1α (HIF1α), and interleukin-1b (IL-1b) in the colon; facilitated the alternative activation of macrophages (Mφs); and inhibited tumor necrosis factor-α (TNFα)-induced reactive oxygen species (ROS) level in intestinal organoids (IOs). Conclusion The JW capsule attenuated the progression of murine colitis by a prompt resolution of inflammation and bloody stool and by re-establishing a microbiome profile that favors re-epithelization and prevents carcinogenesis.

The pharmacological validation of the Xiao-Jian-Zhong formula against ulcerative colitis by network pharmacology integrated with metabolomics

Ethnopharmacological relevanceInflammatory bowel disease (IBD) is pathologically characterized by an immune response accommodative insufficiency and dysbiosis accompanied by persistent epithelial barrier dysfunction, and is divided into ulcerative colitis (UC) and Crohn's disease (CD). Its progression increases the susceptibility to colitis-associated cancer (CAC), as well as other complications. The Xiao-Jian-Zhong (XJZ) formula has a historical application in the clinic to combat gastrointestinal disorders. Aim of the studyThe investigation aimed to explore the molecular and cellular mechanisms of XJZ. Materials and methodsDextran sodium sulfate (DSS) was diluted in drinking water and given to mice for a week to establish murine models of experimental colitis, and the XJZ solution was administered for two weeks. Network pharmacology analysis and weighted gene co-expression network analysis (WGCNA) were utilized to predict the therapeutic role of XJZ against UC and CAC. 16S rRNA sequencing and untargeted metabolomics were conducted utilizing murine feces to examine the changes in the microbiome profile. Biochemical experiments were conducted to confirm the predicted functions. ResultsXJZ treatment markedly attenuated DSS-induced experimental colitis progression, and the targets were enriched in inflammation, infection, and tumorigenesis, predicted by network pharmacology analysis. Based on The Cancer Genome Atlas (TCGA) database, the XJZ-targets were related to the survival probability in patients with colorectal cancer, underlying a potential therapeutic value in cancer intervention. Moreover, the XJZ therapy successfully rescued the decreased richness and diversity of microbiota, suppressed the potentially pathogenic phenotype of the gut microorganisms, and reversed the declined linoleic acid metabolism and increased cytochrome P450 activity in murine colitis models. Our in-vitro experiments confirmed that the XJZ treatment suppressed Caspase1-dependent pyroptosis and increased peroxisome proliferators-activated receptor-γ(PPAR-γ) expression in the colon, facilitated the alternative activation of macrophages (Mφs), inhibited tumor necrosis factor-α (TNFα)-induced reactive oxygen species (ROS) level in intestinal organoids (IOs), thereby favoring the mucosal healing. ConclusionThe XJZ formula is efficacious for colitis by a prompt resolution of inflammation and dysbiosis, and by re-establishing a microbiome profile that favors re-epithelization, and prevents carcinogenesis.

The Binary Mixtures of Lambda-Cyhalothrin, Chlorfenapyr, and Abamectin, against the House Fly Larvae, Musca domestica L.

The house fly Musca domestica L. is one of the medical and veterinary pests that can develop resistance to different insecticides. Mixing insecticides is a new strategy for accelerating pest control; furthermore, it can overcome insect resistance to insecticides. This study aims to evaluate three insecticides, chlorfenapyr, abamectin, and lambda-cyhalothrin, individually and their binary mixtures against 2nd instar larvae of M. domestica laboratory strain. Chlorfenapyr exhibited the most toxic effect on larvae, followed by abamectin then the lambda-cyhalothrin. The half-lethal concentrations (LC50) values were 3.65, 30.6, and 94.89 ppm, respectively. These results revealed that the high potentiation effect was the mixture of abamectin/chlorfenapyr in all the mixing ratios. In contrast, the tested combination of lambda-cyhalothrin/abamectin showed an antagonism effect at all mixing ratios against house fly larvae. The total protein, esterases, glutathione-S-transferase (GST), and cytochrome P-450 activity were also measured in the current investigation in the larvae treated with chlorfenapyr. Our results indicate that GST may play a role in detoxifying chlorfenapyr in M. domestica larvae. The highest activity of glutathione-S-transferase was achieved in treated larvae with chlorfenapyr, and an increase in cytochrome P-450 activity in the larvae was observed post-treatment with Abamectin/chlorfenapyr.

Mechanisms of Increased Indoxacarb Toxicity in Methoxyfenozide-Resistant Cotton Bollworm Helicoverpa armigera (Lepidoptera: Noctuidae).

Indoxacarb is an important insecticide for the selective control of Helicoverpa armigera. It can be bioactivated to the more effective N-decarbomethoxylated indoxacarb (DCJW) by esterases in pests. It was observed that both field and laboratory selected populations of H. armigera showed negative cross-resistance between indoxacarb and methoxyfenozide. The Handan population exhibited moderate resistance to indoxacarb, but was susceptible to methoxyfenozide; the Baoding and Yishui populations exhibited moderate resistance to methoxyfenozide, but they were susceptible to indoxacarb. Moreover, the toxicity of indoxacarb was enhanced 1.83-fold in the laboratory methoxyfenozide-resistant H. armigera, and susceptibility to methoxyfenozide was increased 2.81-fold in the laboratory indoxacarb-resistant H. armigera. In vivo, DCJW concentrations in the susceptible and methoxyfenozide-selected (laboratory methoxyfenozide-resistant) populations were 4.59- and 4.31-fold greater than in the indoxacarb-resistant Handan population 1 h after dosing. After 2 h, the highest concentrations of DCJW and indoxacarb appeared in the methoxyfenozide-selected population. Meanwhile, increased carboxyl esterase (CarE) and decreased glutathione S-transferase (GST) activities were observed in the methoxyfenozide-selected population. However, the indoxacarb-selected (laboratory indoxacarb-resistant) and Handan populations showed a higher disappearance of indoxacarb and DCJW, and the activity of cytochrome P450 mono-oxygenase in these populations were significantly increased. This study showed that the improved toxicity of indoxacarb, as observed in the methoxyfenozide-selected H. armigera, was correlated with increased CarE activity, decreased GST activity, and the in vivo accumulation of indoxacarb and DCJW. The significantly increased cytochrome P450 activity and higher disappearance of indoxacarb and DCJW in indoxacarb-resistant H. armigera resulted in the decreased toxicity of indoxacarb.

Toxicological and molecular profiling of insecticide resistance in a Brazilian strain of fall armyworm resistant to Bt Cry1 proteins.

BackgroundSpodoptera frugiperda, fall armyworm (FAW) is the major pest of maize in Brazil and has readily acquired field resistance to a broad range of synthetic insecticides and to Bacillus thuringiensis (Bt) insecticidal proteins expressed in important crops. This study aims to understand patterns of cross‐resistance in FAW by investigating the toxicological profile of a Bt‐resistant Brazilian strain (Sf_Des) in comparison to a Bt‐susceptible strain (Sf_Bra).ResultsLaboratory bioassays with 15 active substances of nine mode of action classes revealed that Sf_Des has a medium level of resistance to deltamethrin and chlorpyrifos. Very high cross‐resistance was observed among Cry1 toxins, but high susceptibility against Vip3A. Strain Sf_Des exhibited – depending on the substrate – up to 19‐fold increased cytochrome P450 activity in comparison to Sf_Bra. RNA‐Seq data support a major role of P450 enzymes in the detoxification of insecticides because we detected 85 P450 transcripts upregulated in Sf_Des. Quantitative reverse transcription polymerase chain reaction (RT‐qPCR) analysis confirmed that CYP9A‐like and CYP6B39 are significantly upregulated (>200‐fold) in Sf_Des in comparison to Sf_Bra strain. No target‐site mutation linked to pyrethroid resistance was detected, but mutations in the AChE linked to organophosphate resistance were observed in Sf_Des. A Gene Ontology (GO) analysis of differentially expressed genes (DEG) categorized most of them into the biological process category, involved in oxidation–reduction and metabolic processes.ConclusionOur results indicate that multiple/cross‐resistance mechanisms may have developed in the Sf_Des strain to conventional insecticides and Bt insecticidal proteins. The systematic toxicological analysis presented will help to guide recommendations for an efficient resistance management. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Toxicity of cadmium to wheat seedling roots in the presence of graphene oxide

The effects of cadmium (Cd) on wheat seedlings in the presence of graphene oxide (GO) were investigated. Parameters evaluated include root morphology, microtubule protein content, cytochrome P450 activity, and the microcellular structure of wheat seedlings. Compared with treatments with Cd or GO in isolation, treatments combining GO and Cd inhibited the total root length, total root surface area, average root diameter, and number of root hairs. GO combined with Cd also increased cytochrome P450 activity and reduced tubulin content. Cotransport of GO-loading Cd entered root tissues and was then transported to the mesophyll cells; this, in turn, triggered damage to cellular structures, including the cell membranes and chloroplast, leading to root blockage and reduced respiratory efficiency, decreased effectiveness of water and nutrient absorption, and ultimate inhibition of wheat growth and development. These effects of GO exposure were also concentration-dependent. The results indicated that GO amplified the phytotoxicity of Cd in wheat seedling roots. Given the worldwide exposure of the environment to Cd contamination, careful consideration should be given to the effects of GO in combination with Cd in agricultural management.

Host associated variations in neonicotinoid susceptibility in Bemisia tabaci and correlation with CYTP450

Neonicotinoid susceptibility in five populations of Bemisia tabaci (Gennadius) from two major cotton growing regions of India was evaluated. These included-North region: Sriganganagar (Rajasthan), Ludhiana (Punjab) and New Delhi and Central region: Khandwa (Madhya Pradesh) and Amravati (Maharashtra). To establish a correlation between neonicotinoid susceptibility and populations, LC50 (imidacloprid, thiamethoxam and acetamiprid), and activity of cytochrome P450 were explored. Bioassay revealed that population from Amravati recorded the maximum lethal concentration with least susceptibility to neonicotinoids with LC50 values ranging from 1317.21 to 1448.22 mg/l, whereas New Delhi population was the most susceptible (LC50 values ranging from 328.18 to 552.51 mg/l). This was also evident from level of cytochrome P450 levels. The difference in cytochrome P450 monooxygenase activity was observed to be non-significant. New Delhi population was taken as a unity to compare increase in cytochrome P450 activity with increase being in the range of 1.90 to 2.39 for cotton, 2.12 to 3.11 for brinjal and 1.87 to 3.08 for tomato as a hosts.

Sexually Dimorphic Regulation of EET Synthesis and Metabolism: Roles of Estrogen.

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid via cytochrome P450 (CYP)/epoxygenase and are hydrolyzed by soluble epoxide hydrolase (sEH). Circulating and tissue levels of EETs are controlled by CYP (EET synthesis) and sEH (EET degradation). Therefore, both increases in CYP activity and decreases in sEH expression potentiate EET bioavailability, responses that prevail in the female sex as a function of estrogen. This mini review, based on subtitles listed, briefly summarizes studies focusing specifically on (1) female-specific potentiation of CYP/epoxygenase activity to compensate for the endothelial dysfunction; and (2) estrogen-dependent downregulation of sEH expression, which yields divergent actions in both systemic and pulmonary circulation, respectively.Estrogen-Potentiating EET Synthesis in Response to Endothelial Dysfunction: This section summarizes the current understanding regarding the roles of estrogen in facilitating EET synthesis in response to endothelial dysfunction. In this regard, estrogen recruitment of EET-driven signaling serves as a back-up mechanism, which compensates for NO deficiency to preserve endothelium-dependent vasodilator responses and maintain normal blood pressure.Estrogen-Dependent Downregulation of Ephx2/sEH Expression: This section focuses on molecular mechanisms responsible for the female-specific downregulation of sEH expression.Roles of EETs in Systemic Circulation, as a Function of Estrogen-Dependent Downregulation of sEH: This section summarizes studies conducted on animals that are either deficient in the Ephx2 gene (sEH-KO) or have been treated with sEH inhibitors (sEHIs), and exhibit EET-mediated cardiovascular protections in the cerebral, coronary, skeletal, and splanchnic circulations. In particular, the estrogen-inherent silencing of the Ephx2 gene duplicates the action of sEH deficiency, yielding comparable adaptations in attenuated myogenic vasoconstriction, enhanced shear stress-induced vasodilation, and improved cardiac contractility among female WT mice, male sEH-KO and sEHI-treated mice.Roles of Estrogen-Driven EET Production in Pulmonary Circulation: This section reviews epidemiological and clinical studies that provide the correlation between the polymorphism, or mutation of gene(s) involving estrogen metabolism and female predisposition to pulmonary hypertension, and specifically addresses an intrinsic causation between the estrogen-dependent downregulation of Ephx2 gene/sEH expression and female-susceptibility of being pulmonary hypertensive, a topic that has never been explored before. Additionally, the issue of the “estrogen paradox” in the incidence and prognosis of pulmonary hypertension is discussed.

The effect of commercial herbicide exposure on the life history and insecticide resistance phenotypes of the major malaria vector Anopheles arabiensis (Diptera: culicidae)

Herbicides, such as atrazine and glyphosate, are common agrochemicals known to pollute surface ground water. As such, aquatic invertebrates associated with agricultural activities can be exposed to varying doses of these xenobiotics. Anopheles arabiensis, a major malaria vector species in southern Africa, is often closely associated with agricultural activities. This study aimed to examine the effects of larval atrazine or glyphosate exposure on larval and adult life history traits on two laboratory strains of An. arabiensis; one insecticide susceptible (SENN), the other selected for resistance (SENN DDT). Atrazine delayed time to pupation in both strains, but markedly more so in SENN DDT. Glyphosate treatment reduced time to pupation in SENN DDT. Larval atrazine exposure decreased adult longevity in SENN, while both herbicide treatments significantly increased adult longevity in SENN DDT. Larval glyphosate exposure was the more potent enhancer of insecticide tolerance in adult mosquitoes. In SENN DDT, it reduced deltamethrin and malathion-induced mortality, and the LT50 s for these insecticides were increased in association with herbicide exposure. Glyphosate exposure also increased the LT50 s for malathion and deltamethrin in SENN. Exposure to both herbicides had contrasting effects on detoxification enzyme activities. Although both increased cytochrome P450 activity, they had opposite effects on those enzymes involved in reactive oxygen species detoxification. Glyphosate decreased glutathione S-transferase activity, but increased catalase activity with atrazine having the opposite effect. This study demonstrates that larval exposure to the herbicides atrazine and glyphosate can affect the insecticide susceptibilities and life history traits of epidemiological importance in An. arabiensis, with glyphosate being the more potent effector of insecticide resistance.

Exposure Through Runoff and Ground Water Contamination Differentially Impact Behavior and Physiology of Crustaceans in Fluvial Systems.

Chemical pollutants enter aquatic systems through numerous pathways (e.g., surface runoff and ground water contamination), thus associating these contaminant sources with varying hydrodynamic environments. The hydrodynamic environment shapes the temporal and spatial distribution of chemical contaminants through turbulent mixing. The differential dispersal of contaminants is not commonly addressed in ecotoxicological studies and may have varying implications for organism health. The purpose of this study is to understand how differing routes of exposure to atrazine alter social behaviors and physiological responses of aquatic organisms. This study used agonistic encounters in crayfish Orconectes virilis as a behavioral assay to investigate impact of sublethal concentrations of atrazine (0, 40, 80, and 160µg/L) delivered by methods mimicking ground water and surface runoff influx into flow-through exposure arenas for a total of 23h. Each experimental animal participated in a dyadic fight trial with anunexposed opponent. Fight duration and intensity were analyzed. Experimental crayfish hepatopancreas and abdominal muscle tissue samples were analyzed for cytochrome P450 and acetylcholinesterase levels to discern mechanism of detoxification and mode of action of atrazine. Atrazine delivered via runoff decreased crayfish overall fight intensity and contrastingly ground water delivery increased overall fight intensity. The behavioral differences were mirrored by increases in cytochrome P450 activity, whereas no differences were found in acetylcholinesterase activity. This study demonstrates that method of delivery into fluvial systems has differential effects on both behavior and physiology of organisms and emphasizes the need for the consideration of delivery pathway in ecotoxicological studies and water-impairment standards.

Flubendiamide affects visual and locomotory activities of Drosophila melanogaster for three successive generations (P, F1 and F2).

Flubendiamide is widely used in agricultural fields to exterminate a broad spectrum of pests (lepidopteran insects) by disrupting their muscle function. The main objective of this study was to find the effects of flubendiamide on a non-target organism, Drosophila melanogaster (dipteran insect). In the present study, different sub-lethal concentrations of Flubendiamide caused a significant (P < 0.05) decrease in acetylcholinesterase activity and increase in cytochrome P450 activity in adult D. melanogaster. Phototaxis and climbing behaviours were found to significantly (P < 0.05) alter in exposed flies. The observed alteration in phototaxis and climbing behaviours were not restricted to P generation, but were found to be transmitted to subsequent generations (F1 and F2 generation) that had never been directly exposed to the test chemical during their life time. It is only their predecessors (P generation) who have been affronted with different concentrations of Flubendiamide. Humans and Drosophilids share almost 60% genomic similarity and 75% disease gene resemblance. Moreover, most of the circuits governing the behaviours studied involve the inhibition and excitation of neurotransmitters, which are conserved in humans and flies. Thus, the present findings suggest that chronic flubendiamide exposure might induce alteration in neurotransmission leading to discrepancy in the behavioural responses (vision and flight) in other beneficial insects and insect-dependent organisms.

Determination of metabolic resistance mechanisms in pyrethroid-resistant and fipronil-tolerant brown dog ticks.

Rhipicephalus sanguineus (Latreille) (Ixodida: Ixodidae) is a three-host dog tick found worldwide that is able to complete its' entire lifecycle indoors. Options for the management of R. sanguineus are limited and its' control relies largely on only a few acaricidal active ingredients. Previous studies have confirmed permethrin resistance and fipronil tolerance in R. sanguineus populations, commonly conferred by metabolic detoxification or target site mutations. Herein, five strains of permethrin-resistant and three strains of fipronil-tolerant ticks were evaluated for metabolic resistance using synergists to block metabolic enzymes. Synergist studies were completed with triphenyl phosphate (TPP) for esterase inhibition, piperonyl butoxide (PBO) for cytochrome P450 inhibition, and diethyl maleate (DEM) for glutathione-S-transferase inhibition. Additionally, increased esterase activity was confirmed using gel electrophoresis. The most important metabolic detoxification mechanism in permethrin-resistant ticks was increased esterase activity, followed by increased cytochrome P450 activity. The inhibition of metabolic enzymes did not have a marked impact on fipronil-tolerant tick strains.

PWE-006 The Effects of Smoking on Thiopurine Metabolism: Abstract PWE-006 Table 1

Introduction There is an unpredictable relationship between thiopurine methyl transferase (TPMT) and hypermethylation. It is likely that cytochrome P450 (CYP450) enzymes play a role in the demethylation of methylated mercaptopurine (MMP), altering the MMP:Thioguanine nucleotide (TGN) ratio(Table 1). Smoking has been shown to induce CYP450 enzymes. One study on smoking and olanzapine demonstrated a 6 fold increase in CYP450 activity through smoking. 1 This might suggest that smoking could upregulate the demethylation process, reducing MMP. Methods Patients on thiopurines had their smoking status recorded as they attended the inflammatory bowel disease (IBD) clinic. Retrospective data on demographics was collected (Table 1). Only measurable TGNs after 6 weeks of therapy were used for analysis. Metabolites measured whilst on allopurinol were excluded. Average TGNs over the treatment period, MMP and MMP:TGN ratios were compared between smokers and non-smokers. Hypermethylators were defined as any patient with an MMP:TGN ratio of ≥11 during their treatment Results 230 patients were analysed(117 males). Table 1 shows comparison in demographics between smokers and non-smokers Mean MMP for smokers was 1683(SD 2565, median 840) versus 2041 in non-smokers(SD 2401, median 1340)(P = 0.713). Mean MeMP:TGN ratio for smokers was 6.15 (SD 9.06, median 2.22) versus 7.81 in non-smokers (SD 8.01, median 4.70)(P = 0.593). Conclusion There were more hypermethylators in the non-smoking cohort than in the smoking cohort (42.7% versus 28.3%(P Reference 1 Carrillo JA, Herraiz AG, Ramos SI, Gervasini G, Vizcaino S, Benitez J. Role of the smoking-induced cytochrome P450 (CYP)1A2 and polymorphic CYP2D6 in steady-state concentration of olanzapine. J Clin Psychopharmacol . 2003 Apr; 23 (2):119–27. Disclosure of Interest None Declared

Changes in IL-2 and IL-10 during Chronic Administration of Isoniazid, Nevirapine, and Paracetamol in Rats.

The aim of this study was to illustrate the initial subclinical drug-induced liver injury and the associated adaptive immune response by monitoring for the changes in plasma IL-2, IL-10, and some cytochrome P450 activity during chronic administration of nevirapine (NVP), isoniazid (INH), and paracetamol (PAR) in rats without clinical hepatotoxicity. Male Sprague-Dawley (SD) rats were divided into four groups (saline (S), NVP, INH, and PAR) of 25 animals each. The drugs were administered daily for 42 days at therapeutic doses (NVP 200 mg/kg, PAR 500 mg/kg, and INH 20 mg/kg) to the respective groups by oral gavage and five rats per group were sacrificed weekly. All the three drugs induced a subclinical liver injury in the first 2-3 weeks followed by healing, indicating adaption. The liver injury was pathologically similar and was associated with immune stimulation and increased cytochrome P450 activity. NVP- and PAR-induced liver injury lasted up to 14 days while that for INH lasted for 28 days. NVP-induced liver injury was associated with increased IL-2, CD4 count, and CYP3A2 activity, followed by increased IL-10 during the healing phase. In conclusion, the initial drug-induced subclinical liver injury, its spontaneous healing, and the associated adaptive immune response have been demonstrated.

On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro

The in vitro study of liver functions and liver cell specific responses to external stimuli deals with the problem to preserve the in vivo functions of primary hepatocytes. In this study, we used the biochip OrganoPlate(TM) (MIMETAS) that combines different advantages for the cultivation of hepatocytes in vitro: (1) the perfusion flow is achieved without a pump allowing easy handling and placement in the incubator; (2) the phaseguides allow plating of matrix-embedded cells in lanes adjacent to the perfusion flow without physical barrier; and (3) the matrix-embedding ensures indirect contact of the cells to the flow. In order to evaluate the applicability of this biochip for the study of hepatocyte's functions, Matrigel(TM)-embedded HepG2 cells were cultured over three weeks in this biochip and compared to a static Matrigel culture (3D) and a monolayer culture (2D). Chip-cultured cells grew in spheroid-like structures and were characterized by the formation of bile canaliculi and a high viability over 14 days. Hepatocyte-specific physiology was achieved as determined by an increase in albumin production. Improved detoxification metabolism was demonstrated by strongly increased cytochrome P450 activity and urea production. Additionally, chip-cultured cells displayed increased sensitivity to acetaminophen. Altogether, the OrganoPlate seems to be a very useful alternative for the cultivation of hepatocytes, as their behavior was strongly improved over 2D and static 3D cultures and the results were largely comparable and partly superior to the previous reports on biochip-cultured hepatocytes. As for the low technical needs, this platform has the appearance of being highly applicable for further studies of hepatocytes' responses to external stimuli.

Relapsing Brucellosis After Liver Transplantation in a Child

Brucellosis is still an important infectious disease worldwide, especially in the Mediterranean area and in developing countries (1). The treatment of brucellosis is challenging because of relatively high rates of relapse and treatment failure. Obviously, relapse occurs more frequently within the first year if the full course of treatment is not completed (2). It is hypothesized that immunocompromised patients and organ transplant recipients have an increased rate of relapse (3). The hepatotoxicity of drugs used for brucellosis therapy and the drug interactions with immunosuppressive agents are important considerations in the treatment of brucellosis in immunocompromised patients and organ transplant recipients. No guidelines exist in the literature concerning the appropriate regimen and duration of therapy for brucellosis/relapsebrucellosis in these populations. In this report, we describe a child with relapsing brucellosis after liver transplantation, who was treated with two different courses of trimethoprim sulfamethoxazole (TMP-SMZ) and rifampin. A 7-year-old girl with fulminant hepatitis due to Wilson’s disease who had undergone liver transplantation 2 years ago presented to us with a fever that had lasted for more than 2 weeks. Due to the fact that the patient lives in a rural area and had consumed unpasteurized fresh cheese, she was evaluated forbrucellosis. The standard tube agglutination test and immunocapture test were positive at a titer of 1:640. The patientdid not have a history of brucellosis or similar symptoms before transplantation and the donor (mother) had no clinical or serologic evidence of brucellosis. Therefore, we postulated that the immunosuppressive therapy (tacrolimus and mycophenolate mofetil) was the predisposing factor for this infectious disease. The patient was treated with TMP-SMZ and rifampin. After treatment, her clinical and laboratory findings improved dramatically. Tacrolimus levels decreased because of increased cytochrome P450 activity due to rifampin. To prevent acute cellular rejection, the tacrolimus dosage was tripled to maintain normal drug blood levels. TMP-SMZ and rifampin were given for 12 weeks. Brucella serology was negative 4 months after treatment. One year later, the patient was evaluated for another fever of unknown origin. Although the patient was not in contact with infected animals or contaminated milk or milk products, her standard tube agglutination test and immunocapture titers were 1:320 and 1:160, respectively. TMP-SMZ and rifampin therapy were started. The patient’s response to therapy was excellent. Despite the fourfold increase in the dose of tacrolimus, normal blood levels of the drug were not achieved. To prevent acute cellular rejection during brucellosis treatment, mycophenolate mofetil, which had been previously discontinued, was restarted. Antibiotic therapy was used for a periodof 6 months to treat the brucellosis relapse. Despite fluctuations in immunosuppressive therapy, rejection and drug-induced hepatotoxicity were not observed. Antibiotic therapy was successful with no recurrence of infection during 3 years of follow-up. In conclusion, we believe that TMP-SMZ and rifampin therapy seem to be effective and reliable for the treatment of brucellosis in children after liver transplantation and that the length of treatment should be extended compared with that given in nonimmunosuppressed individuals. Ali Islek 1 Ersin Sayar1 Aygen Yilmaz1 Filiz Günseren2 Reha Artan1 1 Department of Pediatric Gastroenterology Akdeniz University Faculty of Medicine Antalya, Turkey 2 Department of Infectious Diseases and Clinical Microbiology Akdeniz University Faculty of Medicine Antalya, Turkey

Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin

Monocyclic aromatic hydrocarbons (MAHs) such as benzene, toluene and xylene are being extensively used for various industrial and household purposes. Exposure to these hydrocarbons, occupationally or non-occupationally, is harmful to organisms including human. Several studies tested for toxicity of benzene, toluene and xylene, and interestingly, only a few studies looked into the attenuation. We used Drosophila model to test the genotoxic and apoptotic potential of these compounds and subsequently evaluated the efficiency of two phytochemicals, namely, quercetin and curcumin in attenuating test chemical induced toxicity. We exposed third instar larvae of wild type Drosophila melanogaster (Oregon R +) to 1.0–100.0 mM benzene, toluene or xylene, individually, for 12, 24 and 48 h and examined their apoptotic and genotoxic potential. We observed significantly ( P < 0.001) increased apoptotic markers and genotoxicity in a concentration- and time-dependent manner in organisms exposed to benzene, toluene or xylene. We also observed significantly ( P < 0.001) increased cytochrome P450 activity in larvae exposed to test chemicals and this was significantly reduced in the presence of 3′,4′-dimethoxyflavone, a known Aryl hydrocarbon receptor (AhR) blocker. Interestingly, we observed a significant reduction in cytochrome P450 activity, GST levels, oxidative stress parameters, genotoxic and apoptotic endpoints when organisms were exposed simultaneously to test chemical along with quercetin or curcumin. The study further suggests the suitability of D. melanogaster as an alternate animal model for toxicological studies involving benzene, toluene and xylene and its potential in studying the protective role(s) of phytochemicals.

The Effect of Selective Inhibition of Inducible Nitric Oxide Synthase on Cytochrome P450 After Liver Transplantation in a Rat Model

Background Activity levels of cytochrome P450 (CYP) provide markers for liver function and graft rejection episodes after orthotopic liver transplantation (OLT). Some in vitro studies have shown decreased CYP activation of inducible nitric oxide synthase (iNOS) in rejecting liver grafts. The aim of this study was to evaluate CYP isoenzyme activity changes in vivo and to examine histopathologic aspects during inhibition of iNOS after treatment with aminoguanidine (AG) using OLT in the rat. Materials and Methods Thirty DA-(RT1av1) rats that served as donors and LEWIS-(RT 1) rats as recipients were divided into three groups: group I (controls, syngeneic rats; n = 6), group II (allogeneic rats without immunosupression; n = 11), and group III (allogeneic rats with AG treatment; n = 13). On postoperative days 5, 8, and 10 we performed laboratory investigations and liver biopsies for histopathologic investigations. Results On postoperative day 5, activities of CYP-1A1 and -3A4 were significantly lower ( P = .022) in group III and the activity of CYP-1A2 higher ( P < .05) compared with group II. At postoperative days 8 and 10, the activities of all CYP isoenzymes were significant higher in AG-treated rats (group III) in contrast with group II after allogeneic OLT without immunosuppression. Histopathologic findings revealed less distinct rejection signs in group III specimens after AG treatment compared with group II. Conclusion Summarizing our results, we concluded that AG treatment led to increased CYP activity and less distinction of graft rejection after OLT in rats.

Overexpression of cerebral and hepatic cytochrome P450s alters behavioral activity of rat offspring following prenatal exposure to lindane

Oral administration of different doses (0.0625, 0.125 or 0.25 mg/kg corresponding to 1/1400th, 1/700th or 1/350th of LD 50) of lindane to the pregnant Wistar rats from gestation days 5 to 21 were found to produce a dose-dependent increase in the activity of cytochrome P450 (CYP)-dependent 7-ethoxyresorufin- O-deethylase (EROD), 7-pentoxyresorufin- O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA- d) in brain and liver of offspring postnatally at 3 weeks. The increase in the activity of CYP monooxygenases was found to be associated with the increase in the mRNA and protein expression of xenobiotic metabolizing CYP1A, 2B and 2E1 isoenzymes in the brain and liver of offspring. Dose-dependent alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 3 weeks have suggested that increase in CYP activity may possibly lead to the formation of metabolites to the levels that may be sufficient to alter the behavioral activity of the offspring. Interestingly, the inductive effect on cerebral and hepatic CYPs was found to persist postnatally up to 6 weeks in the offspring at the relatively higher doses (0.125 and 0.25 mg/kg) of lindane and up to 9 weeks at the highest dose (0.25 mg/kg), though the magnitude of induction was less than that observed at 3 weeks. Alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 6 and 9 weeks, though significant only in the offspring at 3 and 6-week of age, have further indicated that due to the reduced activity of the CYPs during the ontogeny, lindane and its metabolites may not be effectively cleared from the brain. The data suggest that low dose prenatal exposure to the pesticide has the potential to produce overexpression of xenobiotic metabolizing CYPs in brain and liver of the offspring which may account for the behavioral changes observed in the offspring.