Academic literature on the topic 'Beta-naphthoflavone'

Primary cultures of rat hepatocytes were exposed to phenobarbitone, clofibric acid, beta-naphthoflavone, isosafrole or dexamethasone for 3 days, and the induction of several cytochrome P-450 isoenzymes was demonstrated by increased catalytic activity, by Western blotting and by immunocytochemistry. The profiles of isoenzymes induced in vitro were compared with those induced in liver microsomes of rats dosed with the same agents. Clofibric acid, an agent which has not been thoroughly investigated previously, was shown to induce both in vivo and in vitro several P-450 isoenzymes normally inducible by phenobarbitone (PB1a, PB3a and PB3b) or steroids (PB2c). Immunocytochemical studies demonstrated that the inducible isoenzymes of cytochrome P-450 are not distributed evenly throughout the hepatocyte population, and increasing concentrations of phenobarbitone or beta-naphthoflavone in the medium results in an increasing proportion of ‘induced’ cells. However, whereas maximal concentrations of beta-naphthoflavone resulted in virtually all cells containing induced levels of MC1b, a maximal concentration of phenobarbitone resulted in only 30% of the cells containing induced levels of PB3a/PB3b. These results are discussed in relation to the heterogeneous distribution and induction of cytochrome P-450 in the intact liver.

Hydroxylations of pregnenolone (PREG) at the 7 alpha- and 7 beta-positions have been reported in numerous murine tissues and organs and responsible cytochrome P450 (CYP) species await identification. Using thin layer chromatography and gas chromatography-mass spectrometry, we report identification of 7 alpha-hydroxy-PREG and 7 beta-hydroxy-PREG metabolites produced in mouse brain microsome digests and kinetic studies of their production with apparent KM values of 0.5 +/- 0.1 microM and 5.1 +/- 0.6 microM for 7 alpha- and 7 beta-hydroxylation respectively. Investigation of CYP inhibitors and of steroid hormone effects on both 7 alpha- and 7 beta-hydroxylations of PREG showed that: (i) different CYP were involved in 7 alpha- and 7 beta-hydroxylation of PREG because solely 7 alpha-hydroxylation was extensively inhibited by metyrapone, alpha-naphthoflavone, ketoconazole and 3 beta-hydroxysteroids, (ii) CYP 1A2, 2D6, 2B1 and 2B11 were not responsible for 7 alpha- and 7 beta-hydroxylation of PREG because respective specific inhibitors furafylline, quinidine and chloramphenicol triggered no inhibition, (iii) CYP 1A1 was responsible for only part of the 7 beta-hydroxylation of PREG because use of alpha-naphthoflavone, which inhibits specifically CYP 1A1, did not suppress entirely 7 beta-hydroxylation, while ketoconazole, metyrapone and antipyrine, which do not inhibit CYP 1A1, decreased part of the 7 beta-hydroxylation, (iv) 7 alpha-hydroxylation of PREG may be shared with other 3 beta-hydroxysteroids such as isoandrosterone and 5-androstene-3 beta,17 beta-diol which were strong inhibitors, but not with dehydroepiandrosterone which was a non-competitive inhibitor as weak as 3-oxosteroids, and (v) 7 beta-hydroxylation of PREG was not markedly changed by other steroids. Taken together, these findings will be of use for identification of the CYP species responsible for 7 alpha- and 7 beta-hydroxylation of PREG and for studies of their activities in brain.

We report for the first time that beta-naphthoflavone (BNF) abolishes ACTH stimulation of cortisol production in rainbow trout (Oncorhynchus mykiss). There was significantly higher hepatic cytochrome P450 content and ethoxyresorufin O-de-ethylase and uridine-5'-diphosphoglucuronic acid transferase activities in BNF-treated fish than in sham-treated controls. BNF did not significantly affect either plasma turnover or tissue distribution of [3H]cortisol-derived radioactivity. Hepatic membrane fluidity and hepatocyte capacity for cortisol uptake were not altered by BNF as compared with the sham-treated fish. These results taken together suggest that BNF does not affect cortisol-clearance mechanisms in trout. A 3 min handling disturbance period elicited a plasma cortisol response in the sham-treated fish; however, the response in the BNF-treated fish was muted and significantly lower than in the sham fish. This in vivo response corroborates the lack of interrenal sensitivity to ACTH in vitro in the BNF-treated fish, suggesting that BNF affects the ACTH pathway in trout. Our results suggest the possibility that cytochrome P450-inducing compounds may affect cortisol dynamics by decreasing interrenal responsiveness to ACTH stimulation in fish, thereby impairing the physiological responses that are necessary for the animal to cope with the stressor.

Abstract AhR (Aryl hydrocarbon Receptor) is a cytosolic transcription factor functioning as a sensor for environmental toxins. Recently, AhR has been suggested to have a cardinal role in Th17 biology. Although AhR is not a direct regulatory factor for Th17 differentiation its activation is required for the functional differentiation of Th17 cells, and production IL-22. To study the role of AhR in T cell differentiation we have used selective ligands of AhR in an in vitro assay and found that AhR agonist beta-naphthoflavone enhances IL-17 production whereas an AhR inhibitor suppresses it, both at the level of transcription and translation. The Th2 cytokine IL-4 was suppressed by beta-naphthoflavone and induced by the inhibitor and the Th1 cytokine IFNγ was inhibited by both the compounds. To further investigate the role of AhR in Th17 biology we used a well established Th17 dominated murine model of hypersensitivity pneumonitis mediated by a thermophilic actinomycete Staphylopolyspora rectivirgula (SR). Analysis of the cytokine transcripts from the SR exposed lungs revealed that IL-17 was not only expressed in WT mice but was more highly expressed in Ahr-null mice. These data suggest that the AhR may regulate cytokine production by other T cell subsets, and that the requirement for the AhR in IL-17 expression may be restricted to T cells. Further work is required to determine the role of the AhR in the innate immune cells, which are potential sources of IL-17.

An inhibitor of hepatic uroporphyrinogen decarboxylase (EC 4.1.1.37) was demonstrated in heat-treated extracts of livers from C57BL/10ScSn mice with iron overload after a single dose (100 mg/kg; 350 mumol/kg) of hexachlorobenzene (HCB). Inhibition was not due to accumulated uroporphyrin since this could be removed by a SEP-PAK C18 cartridge without affecting inhibitor activity. The presence of the inhibitor could be first demonstrated 2 weeks after mice received HCB and before major elevation of hepatic porphyrin levels. Maximum inhibitory potential was reached at about 8 weeks and was still detected 25 weeks after the chemical, thus paralleling the depression of enzyme activity reported previously [Smith, Francis, Kay, Greig & Stewart (1986) Biochem. J. 238, 871-878]. The inhibitor was not detected following treatment of mice with either iron or HCB alone or after the decarboxylase activity was destroyed in vitro by the combination of uroporphyrin and light. The formation of the inhibitor by inbred mouse strains nominally Ah-responsive (C57BL/6J, C57BL/10ScSn, BALB/c, C3H/HeJ, CBA/J and A/J) and Ah-nonresponsive (SWR, AKR, 129, SJL, LP and DBA/2) did not correlate fully with their reported Ah-phenotype. There was a correlation amongst the Ah-responsive strains only, with hepatic ethoxyphenoxazone de-ethylase activity induced in parallel experiments by treatment with beta-naphthoflavone. De-ethylase activity induced by HCB, however, was considerably less than that with beta-naphthoflavone, which has not been reported as porphyrogenic. Other polyhalogenated chemicals, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,2′,3′,4′-hexachlorobiphenyl and hexabromobenzene, also caused the formation of the inhibitor of uroporphyrinogen decarboxylase.

The circadian clock system is essential for mammals to adapt to environmental conditions such as light-dark cycles and to manage the optimal timing for cyclical physiological processes, including sleep-wakefulness, fasting-feeding and multiple aspects of metabolism. The circadian timing system is arranged in hierarchical fashion, with the master clock in the suprachiasmatic nucleus (SCN) of the hypothalamus, acting as the pace-maker and maintaining synchrony among clocks found in every organ system throughout the body. The core molecular clock consists of two interconnected transcriptional-translational feedback loops comprising core clock components: Brain and Muscle Arnt-Like protein1 (BMAL1), Circadian Locomoter Output Cycles Kaput (CLOCK), Period (PER), Cryptochrome (CRY), Nuclear Receptor family1 D1 (REV-ERB) and Retinoic acid-related Orphan Receptor (ROR). Circadian clock disruptions, through environmental changes to light-dark cycles or through genetic modification of core clock genes cause metabolic disturbances. Aryl hydrocarbon receptor (AhR), also known as the dioxin receptor, mediates systemic metabolism and toxicity of a range of environmental contaminants. Epidemiological studies have established a positive correlation between exposure to dioxins and other synthetic organic chemicals and metabolic diseases such as diabetes and dyslipidemia. Animal research have supported these findings by showing that AhR activation has detrimental effects on glucose and lipid homeostasis. Mechanisms for AhR-mediated metabolic dysfunction remain unknown. Coincidently, both AhR and many core clock components, for example BMAL1 and CLOCK, belong to the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) domain family. Previous studies have linked AhR signaling to circadian rhythm. Importantly, activation of the AhR can impair transcriptional activity of the CLOCK: BMAL1 heterodimer in cultured cells. However, because the AhR is differentially expressed among the body’s tissues, its activation may have distinctive, tissue-specific effects on the hierarchical circadian clock oscillators in vivo, which have not been investigated. Therefore, this dissertation is designed to examine the short-term and long-term effects of AhR activation on circadian clocks and downstream clock-regulated metabolic pathways. Specifically, this dissertation is aimed to explore how acute and chronic activation of AhR affects rhythmic aspects of behavior, as well as clock-controlled glucose and lipid metabolism. In the acute AhR activation model, a single dose of the synthetic AhR agonist, β-Naphthoflavone (BNF), was administered to C57Bl/6J wild type mice. Circadian behavior was monitored before and after acute AhR activation. Circadian expression of core clock genes, as well as key metabolic genes in the liver, skeletal muscle and adipose tissue were examined. Compared to the vehicle group, BNF-treated mice displayed a transient loss of behavioral rhythmicity and delayed activity onset, which suggest that acute activation of AhR acts directly on the central clock, the suprachiasmatic nucleus of the hypothalamus. In contrast, circadian oscillations of core clock genes were not eliminated in the peripheral tissues (liver, skeletal muscle and adipose tissue), but changes were observed in their rhythmic amplitude or phase. Rhythms of key enzymes related to glucose and lipid metabolic pathways in the liver and adipose were decreased while those in the skeletal muscle were increased. These results indicate that acute AhR activation affects the central clock and peripheral clock differently. Moreover, acute AhR activation significantly dampened the rhythm of genes involved in lipogenesis, lipolysis and lipid storage. In the chronic AhR activation model, C57Bl6/J mice were exposed to BNF for a month to explore whether long-term AhR activation can cause bigger disruption of circadian clocks and lead to metabolic dysfunction in vivo. Unexpectedly, general circadian behavior was maintained although after each dose of BNF there was a consistent, transient loss of behavioral rhythmicity and significant phase delay (about 30 minutes) in BNF-treated mice. Liver and skeletal muscle clocks were not significantly altered after 4 doses of BNF, and the in-phase oscillations of core clock genes in liver and skeletal muscle suggested a functional SCN as well as the two peripheral clocks. However, the adipose clock was significantly disrupted. Altered clock-regulated rhythms in lipid metabolism genes are associated with impaired lipid storage functions in white adipose tissues and deregulated plasma lipids in BNF-treated mice. The results of acute and chronic AhR activation support a significant interaction of AhR with the circadian clock system. Although future studies are needed to elucidate how AhR signaling specifically interacts with the clock in different cell types, the current research establishes a model for studying the crosstalk between AhR and circadian rhythm and provides new perspectives into the mechanisms of metabolic diseases correlated with exposure to synthetic organic chemicals.

2012/2013
Abstract Hyperbilirubinemia is the most common clinical diagnosis during neona- tal life. The neonatal jaundice may be physiological without any clinical consequence, or can lead to an acute form of bilirubin en- cephalopathy with minimal neurological damage, or to more severe and dangerous condition called kernicterus (permanent neurological sequelae). The Gunn rat is one of the models in which bilirubin encephalopathy was studied. It was shown that jaundiced newborn animals display an hepatic compensatory mechanism for the incapacity to eliminate bilirubin (Ugt1A1 mutant enzyme) by upregulation of cyotochrome P450 enzymes (Cyp) 1A1 and 1A2 compared with their not jaun- diced littermates. In addition, their expression in brain was selec- tively induced (cortex higher and early vs. cerebellum lower and late induction) by sudden increase of bilirubin content. Cyotochrome P450 enzymes are the main enzymes involved in the detoxification of drug and endogenous compounds. In liver their role in bilirubin oxidation has been deeply demonstrated. In brain, there are many works that analyzed Cyps expression and induction, but their role in bilirubin oxidation has never been evaluated till now. This thesis focused in; 1) analyze the inducibility of Cyps enzymes, by a known inducer, βNF in cortex and cerebellum astrocytes; 2) Select the optimal conditions to induce specifically each of them; 3) analyze their activity and 4) evaluate their capacity to oxidize bilirubin; 5) Last but not least, assess the protective effect of Cyps induction by challenging the cells with high concentrations of bilirubin.We demonstrated that astrocytes Cyps were inducible (both mRNA and activity) by βNF and this induction varies depending on the brain region and among the Cyps. To explain the differences we hypoth- esized the presence of: i) a different protein isoforms (discarded for Cyp1A1) ii) different maturation stage of astrocytes in the two brain regions (P2 rats: Cll is less developed than Cx), iii) differential regu- latory mechanisms. Our work showed that in Cx astrocytes Cyp1A1 induction (mRNA and activity) was an early event (6h), requiring low concentration of the drug, making this approach theoretically possible in vivo. On the other hand, to reach the higher levels of Cyp1A1 (mRNA and activ- ity) in Cll we needed 24h. In both cases Cyp1A1 was able to oxidize bilirubin producing an increase in viability only after TCB addition. The Cyp1A2 was the major catalyst of bilirubin degradation (with- out the need of TCB), but its modulation was more difficult to be achieved. In addition, while Cyp1A2 modulation increased bilirubin oxidation in cortex (also reflected in an increment in viability), in cerebellum we noticed a slight reduction in bilirubin clearance (no improvements in viability). All this data could lead to the basis of the cerebellum susceptibility to UCB. The observation that the TCB addition in Cyp1A1 astrocytes increases bilirubin oxidation and via- bility suggest the possibility to induce Cll resistance toward bilirubin neurotoxicity by Cyps modulation and uncoupling. --------------------------------------------- Riassunto Una delle condizioni neonatali pi`u comuni `e l’ittero. Esso rappresenta l’aumento fisiologico della bilirubina nel sangue e tessuti. Tuttavia esso pu`o divenire una condizione patologica (encefalopatia da biliru- bina), caratterizzata da un danno neurologico minimo, o condurre a una condizione severa e pericolosa per il neonato chiamata kernit- tero (danni permanenti), in caso di una esposizione prolungata ad alti livelli del pigmento. Attualmente, la patologia da iperbilirubinemia rappresenta la principale causa di riammissione in ospedale nel primo mese di vita. Per il danno neurologico da bilirubina esiste un modello animale, il ratto Gunn, che presenta la stessa mutazione genica dei pazienti Crigler-Najjar I. Come nella condizione umana, esso manca della uridin di fosfo glucuronisil tranferasi 1a1 (Ugt1A1), enzima epatico deputato alla coniugazione della bilirubina. Tuttavia l’animale ri- esce a compensare la mancanza mantenendo bassi i livelli serici di bilirubina, grazie ad una incrementata attivit`a delle citocromo P450 mono-ossigenasi epatiche (Cyps, Cyp1A1 and Cyp1A2). Tali enzimi riducono la concentrazione del pigmento attraverso la sua ossidazione. ` E stato supposto che questo meccanismo possa essere alla base anche della capacit`a di specifiche aree del cervello (cortex e collicoli superiori, vs. i danneggiati cervelletto, ippocampo e collicoli inferiori) di ridurre la concentrazione del pigmento, limitando/impedendo il danno neu- rologico associato. Sebbene il loro ruolo, i meccanismi ed i prodotti della loro attivit`a di clearance della bilirubina siano stati ampiamente dimostrati in fegato, in cervello non sono ancora disponibili evidenze funzionali della loro capacit`a di ossidare la bilirubina. L’obiettivo di questa tesi `e stato di analizzare 1) la modulazione delle Cyps cerebrali (brain Cyp: bCyp) utilizzando un noto induttore, βNF; 2) selezionare le condizione ottimali per una induzione selettiva di cias- cuna bCyp; 3) allo scopo di valutare la loro attivit`a (EROD/MROD);4) e la capacit`a specifica di ossidare bilirubina; 5) conferendo pro- tezione (aumento vitalit`a) alle culture primarie di astrociti (da cortex e cervelletto) esposte a dosi tossiche di bilirubina. Questo studio ha dimostrato la inducibilit`a delle bCyps (mRNA e attivit`a) tramite βNF. Abbiamo evidenziato una modulazione dipen- dente della regione cerebrale da cui sono state prodotte le cellule, in aggiunta ad un comportamento differenziale delle Cyps tra loro. Pos- sibili spiegazioni possono essere: i) la presenza di diverse isoforme tra le regioni (ipotesi scartata in seguito ad analisi tramite western blot); ii) diversa presenza delle vie di segnalazione alla base dei meccanismi di modulazione, o, iii) una diversa risposta dovuta al diverso stadio sviluppo degli astrociti preparati dalle due regioni (ratti P2: il Cll `e meno sviluppato del Cx). Inoltre, abbiamo documentato una precoce (6h) inducibilit`a della Cyp1A1 nei astrociti dal Cx, che inoltre necessit`a di minori concen- trazioni del principio. Tali caratteristiche suggeriscono che la Cyp1A1 sia pi`u facilmente modulabile in vivo. Tuttavia, per ottenere una buona modulazione nel cervelletto (regione pi`u sensibile al danno), bisogna mantenere la concentrazione dell’ induttore per un tempo lungo (24h). In entrambe le regioni, un incremento significativo della capacit`a di ossidare bilirubina, migliorando la vitalit`a delle cellule, `e stato ottenuto solo dopo il co-trattamento con TCB (disaccoppia- mento). La Cyp1A2 `e stata significativamente modulata solo negli astrociti da Cx (24h), in cui abbiamo evidenziato un chiara capacit`a dell’enzima di ossidare la bilirubina e migliorare la vitalit`a in assenza di TCB. I risultati ottenuti permettono di comprendere la suscettibilit`a differenziale (tra regioni del cervello) tipica di questa patologia. La capacit`a di indurre Cyp1A1 e renderli pronti a ossidare la bilirubina nei astrociti da Cll (regione pi`u danneggiata) e Cyp1A1/Cyp1A2 in Cx e oltre migliorando la viabilit`a di questi suggeriscono la possibilit`a di conferire resistenza a la tossicit`a da bilirubina.
XXVI Ciclo
1986