Effects Of Β-naphthoflavone Research Articles

A novel contact assay for testing aryl hydrocarbon receptor (AhR)-mediated toxicity of chemicals and whole sediments in zebrafish (Danio rerio) embryos.

The European Water Framework Directive aims to achieve a good ecological and chemical status in surface waters until 2015. Sediment toxicology plays a major role in this intention as sediments can act as a secondary source of pollution. In order to fulfill this legal obligation, there is an urgent need to develop whole-sediment exposure protocols, since sediment contact assays represent the most realistic scenario to simulate in situ exposure conditions. Therefore, in the present study, a vertebrate sediment contact assay to determine aryl hydrocarbon receptor (AhR)-mediated activity of particle-bound pollutants was developed. Furthermore, the activity and the expression of the CYP1 family in early life stages of zebrafish after exposure to freeze-dried sediment samples were investigated. In order to validate the developed protocol, effects of β-naphthoflavone and three selected sediment on zebrafish embryos were investigated. Results documented clearly AhR-mediated toxicity after exposure to β-naphthoflavone (β-NF) and to the sediment from the Vering canal. Upregulation of mRNA levels was observed for all investigated sediment samples. The highest levels of all investigated cyp genes (cyp1a, cyp1b1, cyp1c1, and cyp1c2) were recorded after exposure to the sediment sample of the Vering canal. In conclusion, the newly developed sediment contact assay can be recommended for the investigation of dioxin-like activities of single substances and the bioavailable fraction of complex environmental samples. Moreover, the exposure of whole zebrafish embryos to native (freeze-dried) sediment samples represents a highly realistic and ecologically relevant exposure scenario.

The effect of β-naphthoflavone on the metabolism of amiodarone by hepatic and extra-hepatic microsomes

Amiodarone is a potent antiarrhythmic drug with several limiting side effects, some of which have been correlated with increased levels of its more toxic metabolite, desethylamiodarone. Elevated serum desethylamiodarone to amiodarone ratios are associated with a risk of amiodarone-induced pulmonary toxicity. Polycyclic aromatic hydrocarbons such as β-naphthoflavone are known to increase desethylamiodarone levels in rat in vivo. In this article we investigated if this increase was solely due to increased formation as a result of cytochrome P450 (CYP) 1A1 and 1A2 induction in different rat hepatic and extra-hepatic tissues. Additionally, the effect of amiodarone treatment on CYP1A1 and 1A2 gene expression and activity was investigated. In rats, β-naphthoflavone was found to increase desethylamiodarone forming activity in lung and kidney microsomes. Amiodarone increased β-naphthoflavone mediated induction of CYP1A1 gene expression in liver, lung and kidney. However, there was no significant change in CYP1A activity. As expected, the data indicated that the increase in desethylamiodarone levels in vivo was partly due to increased formation through CYP1A1 induction, although increased formation was only evident in some extra-hepatic tissues. Amiodarone treatment did not affect basal or induced CYP1A activity.

Effect of β-naphthoflavone on AhR-regulated genes (CYP1A1, 1A2, 1B1, 2S1, Nrf2, and GST) and antioxidant enzymes in various brain regions of pig

The constitutive and inducible expression of aryl hydrocarbon receptor (AhR) and of the AhR-regulated genes coding for CYP1A1, CYP1A2, CYP1B1, CYP2S1, and Nrf2 was investigated by real-time or traditional PCR in cerebral areas (cortex, cerebellum, midbrain, and hippocampus), blood–brain interfaces (meninges and brain microvessels) and liver obtained from control pigs and from pigs treated with β-naphthoflavone (βNF), a potent AhR agonist. The enzymatic activities of ethoxyresorufin-O-deethylase (EROD), and methoxyresorufin-O-deethylase (MEROD), marker for CYP1A1 and CYP1A2, the GST and various antioxidant enzymes (catalase, superoxide dismutase, GSSG-reductase, and GSH-peroxidase) were also determined in the same CNS regions. The AhR, CYP1A1, CYP1A2, CYP1B1, Nrf2 mRNAs were detected, although at different extent, in all the CNS regions, while CYP2S1 mRNA was detected only in midbrain. In the blood–brain interfaces, the constitutive basal expression of AhR and CYP1A1 was comparable to the hepatic one and even higher for CYP1B1 and Nrf2. The treatment with βNF determined the induction of CYP1A1 and 1B1 (but not of AhR, CYP1A2, and Nrf2) mRNA levels in various CNS areas; notably, CYP1A1 mRNA was increased to about 300-fold in the microvessels. The analysis of enzymatic activities revealed that EROD, but not MEROD, was induced in microsomes but not in mitochondria of all the CNS areas. However, the mitochondrial EROD activities were comparable (in midbrain, meninges) or higher (in cortex, cerebellum, hippocampus) than the microsomal ones, suggesting an important metabolic function of CYP1A1 in this subcellular localization. The activities of GST and antioxidant enzymes were detected in all CNS tissues, with levels lower than the hepatic ones, but found quite evenly distributed and marginally affected by βNF treatment. The high expression of metabolic enzymes found in blood–brain interfaces could represent a very important defence toward toxins of CNS.

Effects of β-naphthoflavone, phenobarbital and dichlobenil on the drug-metabolizing system of liver and nasal mucosa of Italian water frogs

In this study, we have examined the presence and inducibility of phase I and II drug-metabolizing enzymes in the liver and nasal mucosa of Italian water frogs of control and pretreated with β-naphthoflavone, phenobarbital and dichlobenil by using typical substrates for these enzymes along with polyclonal antibodies mainly raised against mammalian enzymes. The CYP content and various monooxygenase and phase II enzyme activities in the liver of this frog were found similar, when reported, to those of largely aquatic and semiaquatic frogs. The treatment with β-naphthoflavone resulted in an induction in the liver of a CYP1A and the induction was manifested by (a) immunoblot analysis using anti-rat CYP1A1, (b) an increase of CYP1A-mediated methoxyresorufin- O-demethylase and ethoxyresorufin- O-deethylase activities. The treatments with both phenobarbital and dichlobenil did not produce in the liver any effect on the assayed enzymes. When the nasal mucosa of water frogs was analyzed, various monooxygenase and phase II enzymatic activities, generally comparable to those of liver, were determined. However, by using antibodies anti-three GST different classes, we found a different reactivity into the cytosol of the two tissues indicating a differential tissue susceptibility to toxic effects of xenobiotics. In the nasal mucosa, a protein immunorelated to CYP2A and monooxygenase activities (i.e. ethoxycoumarin- O-deethylase and coumarin-7-hydroxylase) linked in mammals to this isoform have also been found. The treatment of water frogs with the herbicide dichlobenil decreased both the above-mentioned activities and the immunoreactive CYP2A apoprotein. The pretreatment with metyrapone, a CYP inhibitor, protected the CYP2A apoprotein and its linked activities from toxic effect of dichlobenil indicating a key role of this enzyme in the bioactivation of this herbicide. The findings of the present work suggest that the hepatic CYP1A induction and the nasal CYP2A-like inhibition profiles might provide two potential biomarkers of the Italian water frogs exposure to environmental and aquatic pollutants.

Effects of β-naphthoflavone on the cytochrome P450 system, and phase II enzymes in gilthead seabream ( Sparus aurata)

The effect of β-naphthoflavone (β-NF) on several catalytic activities of cytochrome P450 (CYP) and phase II enzymes putatively controlled by [Ah]-receptor activation in the liver, heart and kidney of gilthead seabream, was investigated. In the liver, β-NF treatment [intraperitoneal injection (i.p.) 50 mg/kg] resulted in an increase of CYP content, immunoreactive CYP 1A and methoxyresorufin- O-demethylase (MEROD), pentoxyresorufin O-depentylase (PROD) and ethoxyresorufin- O-deethylase (EROD) activities. However, β-NF had no effect on any of the hepatic phase II enzymes examined (benzaldehyde dehydrogenase, propionaldehyde dehydrogenase, glutathione S-transferase, UDP-glucuronyl-transferase, DT-diaphorase). Single i.p. injection of 10 mg/kg β-NF showed a maximal induction of CYP 1A-like protein and EROD activity after 3–7 days. CYP 1A and EROD returned to control levels 18-days post-treatment. β-NF injection also caused a rapid increase of a single band size of mRNA recognized by a CYP 1A1 cDNA fragment from sea bass ( Dicentrarchus labrax). Expression of mRNA preceded the increase of EROD activity and declined rapidly by 96 h. Dose–response experiments demonstrated that EROD was significantly enhanced in liver by a single injection of 0.3 mg/kg β-NF and was the most sensitive measurement for CYP 1A-like induction. β-NF treatments also increased the expression of CYP 1A-like protein, mRNA and EROD, but not MEROD and PROD activities in heart and kidney.

Effect of β-naphthoflavone and dimethylbenz[ a]anthracene on apoptosis and HSP70 expression in juvenile channel catfish ( Ictalurus punctatus) ovary

Complex environmental mixtures such as pulp mill effluents and crude oil have been shown to increase ovarian cell apoptosis and affect heat shock protein (HSP) expression in fish. We hypothesize that polyaromatic hydrocarbons (PAH) mediate these effects. To test this hypothesis, we exposed juvenile channel catfish ( Ictalurus punctatus) acutely to the aryl hydrocarbon receptor (AhR) agonists, β-naphthoflavone (BNF; 75 mg/kg) or the model PAH, dimethylbenz[ a]anthracene (DMBA; 50 mg/kg) via intraperitoneal injection. Apoptotic DNA fragmentation and HSP70 expression were determined in ovary and liver. Hepatic cytochrome P450 1A (CYP1A) was significantly induced, confirming that BNF and DMBA had distributed to internal organs and stimulated AhR. At 96 h post-injection, BNF and DMBA significantly increased apoptosis and decreased HSP70 expression in juvenile catfish ovaries. Although primary oocytes underwent the greatest rates of apoptosis compared to early or late vitellogenic follicles in all treatment groups, the cell type undergoing increased rates of apoptosis after BNF or DMBA exposure was not clear using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyUTP nick end labeling (TUNEL). There was a significant negative relationship between expression of HSP70 and apoptosis in juvenile channel catfish ovaries. This differed from liver of these fish which did not exhibit increased apoptosis and instead increased hepatic HSP70 expression at 96 h post-injection. However, DMBA had no effect on apoptosis or HSP70 levels in more reproductively mature juvenile fish that were housed at a lower water temperature. This may be due to a developmental or temperature-dependent component to these responses. We propose that the decrease in ovarian HSP70 expression in response to BNF and DMBA may be causally related to the increase in ovarian cell apoptosis. Further experiments using a full time course, dose–response and methods to confirm that AhR is a direct mediator of these effects are required.

Effects of β-naphthoflavone and cadmium on inducibility of CYT P450 monooxygenases in fresh water fishes

Effects of β-naphthoflavone and cadmium on inducibility of CYT P450 monooxygenases in fresh water fishes

Transplantation of fetal liver tissue suspension into the spleens ofadult syngenic rats: Effects of β-naphthoflavone, Phenobarbital and dexamethasone on cytochrome P450 isoforms expression and on glycogen storage

In the present study, the effect of beta-naphthoflavone (BNF), phenobarbital (PB) and dexamethasone (DEX) on the expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, and on glycogen storage was investigated in intrasplenic liver cell explants in comparison to adult liver. Fetal liver tissue suspensions were transplanted into the spleens of adult male syngenic Fisher inbred rats. Four months after surgery, transplant recipients and age matched controls were orally treated with BNF (1 x 50 mg/kg body weight (b.wt.)), PB (1 x 50 mg/kg b.wt.), DEX (for 3 days 4 mg/kg b.wt. per day), or the respective solvents (dimethylsulfoxide or 0.9% NaCl). The animals were sacrificed 24 (BNF, DEX) or 48 (PB) hours after the last treatment. The livers of both solvent treated transplant recipients and control rats displayed only in few liver lobules a slight P450 1A1, but in all lobules a strong P450 2B1 and 3A2 expression, which was all mainly located in the hepatocytes around the central veins (zone III, according to Rappaport). After BNF administration a P450 1A1 expression was induced in the hepatocytes of the peripheral regions of the liver lobules (zone I, according to Rappaport), whereas the staining of the hepatocytes around the central veins disappeared. Also the staining for P450 2B1 in the hepatocytes of zone III became slightly more pronounced. Following PB treatment the P450 1A1 expression in the hepatocytes of the central regions (zone III), as seen in few lobules after solvent treatment only, was reduced, whereas the staining for P450 2B1 and 3A2 was more pronounced in the hepatocytes of the intermedial and central regions of the liver lobules (zone II and III). DEX treatment diminished P450 1A1 and 2B1 expression within the livers of both transplant recipients and control rats. In contrast, the staining for P450 3A2 was enhanced in all regions of the liver lobules. Transplantation of fetal liver tissue suspensions into the spleens did not influence the inducibility of P450 isoforms expression within the respective livers of the animals. Spleens of control rats displayed no P450 isoforms expression without as well as with induction. In the explant containing spleens, however, similar to normal liver, the transplanted hepatocytes displayed nearly no P450 1A1, but a strong P450 2B1 and 3A2 expression. After BNF treatment a staining for P450 1A1 was induced and also the P450 2B1 expression was slightly more pronounced. PB treatment caused an increase in the staining for P450 2B1 and 3A2 and DEX administration for P450 3A2 within the transplanted hepatocytes. Additionally, after DEX treatment some bile ducts of the explants displayed a slight staining for P450 1A1, 2B1 and 3A2. All hepatocytes within the livers of both solvent treated transplant recipients and control rats displayed a slightly PAS-positive cytoplasma and, in most cases, homogeneously distributed, fine-grained, strongly PAS-stained granules indicating glycogen storage. No regional variance in the glycogen content of the hepatocytes was seen within the liver lobules, but there was a marked difference between the individual hepatocytes of the same lobular region in the extent of glycogen accumulation. The hepatocytes within the explants displayed the same type of glycogen storage as did the adult liver cells. BNF treatment did not display any effect on the glycogen accumulation in livers and intrasplenic liver cell explants. After PB administration, only in livers, but not in the transplants, the glycogen content in the hepatocytes around the central veins was slightly reduced. DEX treatment lead to an excessive storage of fat within the hepatocytes of both livers and spleens. Thus, the glycogen was displaced, leading to a "spoke-wheel" like pattern of glycogen storage. Additionally, within the hepatocytes of both livers and liver cell explants a higher amount of glycogen seemed to be stored and the granules appeared to be more coarse-grained. (ABSTRACT

Effects of β-naphthoflavone on the cytochrome P450 enzyme system in the mature male rainbow trout kidney

Effects of β-naphthoflavone on the cytochrome P450 enzyme system in the mature male rainbow trout kidney

Effect of β-naphthoflavone on multiple cytochrome P450 forms in primary cultures of rainbow trout hepatocytes : JOHN J. STEGEMAN, ∗ MICHAEL J. MILLER,‡ BRUCE R. WOODIN ∗ & JAMES D. BLAIR,‡

Effect of β-naphthoflavone on multiple cytochrome P450 forms in primary cultures of rainbow trout hepatocytes : JOHN J. STEGEMAN, ∗ MICHAEL J. MILLER,‡ BRUCE R. WOODIN ∗ & JAMES D. BLAIR,‡

Non-induction of extrahepatic antipyrine and metronidazole metabolism evaluated from partially hepatectomized rats.

1. The effect of beta-naphthoflavone (BNF), given i.p. (n = 9) and orally (n = 9), on the metabolism of antipyrine and metronidazole was investigated in rats. 2. The clearances of antipyrine and metronidazole were determined on a single saliva sample. The rates of formation of antipyrine and metronidazole metabolites were determined from a 20 h urine sample and saliva clearance. 3. Administration of beta-naphthoflavone i.p. was significantly more effective than oral dosage on the induction of antipyrine and metronidazole metabolism (p < 0.05). 4. The capacity of extrahepatic tissues to metabolize antipyrine and metronidazole was quantitatively assessed in rats with and without pretreatment with beta-naphthoflavone immediately after sham operation or 70% partial hepatectomy (n = 40). 5. Antipyrine and metronidazole clearances correlated with liver weight in induced and non-induced rats. Linear regression of antipyrine and metronidazole clearances did show a non-significant Y-intercept (p > 0.05), indicating a negligible extrahepatic metabolism in both induced and in non-induced rats. 6. From a quantitative point of view this study indicates that induction of extrahepatic cytochrome P450 metabolism of antipyrine and metronidazole is negligible.

Effect of β-naphthoflavone and MCPA on liver and kidney drug-metabolizing enzymes from the carp, Cyprinus carpio

The effects of β-naphthoflavone (β-NF) and a chlorophenoxyacetic acid herbicide (MCPA) on hepatic and renal monooxygenase activities and conjugating enzymes from immature carp ( Cyprinus carpio) were studied. β-NF increased hepatic monooxygenase activities but the patterns of differential induction generally obtained in rat liver microsomes with two series of homologous substrates, alkoxycoumarins and alkylresorufins, were not found to be similar in carp liver microsomes. On the other hand, MCPA caused no changes in oxidative metabolism, with the exception of decreased aryl hydrocarbon hydroxylase activity. Renal activities were not modified by MCPA, while β-NF treatment resulted in marked increases in monooxygenase activities with alkylresorufins as substrates. No changes were found in conjugation activities after treatment with MCPA or β-NF. These results indicate that (a) the herbicide MCPA should have no effect on drug-metabolizing enzymes from carp, and (b) the hepatic and renal monooxygenase activities of carp are responsive to β-NF, allowing their use in monitoring water pollution.

Effect of β-naphthoflavone on the metabolism of aflatoxin B 1 in hamsters

The effect of β-naphthoflavone (BNF) pretreatment of hamsters on the hepatic metabolism of aflatoxin B 1 (AFB 1) has been examined in studies in vitro and in vivo. Pretreatment with BNF not only increased microsomal cytochrome P-450 by 50–80% but also increased microsome-mediated AFB 1 epoxidation as measured by AFB 1-DNA binding 2.6 fold without significantly affecting other hydroxylations. Neither cytosolic GSH S-transferases' activities nor AFB 1-GSH (AFB 1-SG) conjugation were affected. In vivo, hepatic AFB 1-DNA binding was also increased about 3–4-fold. These results in contrast to those observed in the rat indicate that induced species of cytochrome P-450 are primarily responsible for higher epoxidation of AFB 1 in the hamster.

H-2, Ah, and aging: The immune response and the inducibility of P-450 mediated monooxygenase activities, xanthine oxidase, and lipid peroxidation in H-2 congenic mice on C57BL/10, C3H, and a strain backgrounds

The effects of β-naphthoflavone (β-NF, 80 mg/kg i.p. for 2 consecutive days) on P-450-dependent and -independent enzymes, lipid peroxidation and xanthine oxidase were investigated in 9 strains of young (3-month-old) male mice. Three H-2 congenic strains on each of three different genetic backgrounds were studied. The backgrounds were C57BL/10 (abbreviated as B10), C3H, and A strain mice. The reported longevities (weeks) as expressed in 10th decile of survivorship are significantly different among the H-2 congenic strains on each of these backgrounds: it ranges from 155 to 170 weeks in B10, from 138 to 150 in C3H and from 114 to 134 in A background mice. The inducibility of aryl hydrocarbon hydroxylase (AHH) with β-NF was highest in B10, intermediate in C3H/He and non-inducible in other C3H mice and in all mice on the A strain background. Within the B10 background, inducibility of AHH varied widely among mice of different H-2 haplotypes: 549±34 (H-2 k), 360±72 (H-2 b) and 349±47 (H-2 r) percent of the mean control values ( n=5; mean±S.D.), without change in activities of P-450-independent enzymes. In C3H mice the H-2 k haplotype showed inducibility (213±34%), while other haplotypes, specifically H-2 b and H-2 j, did not. β-NF increased the activities of xanthine oxidase in B10 and A background strains, without interbackground differences. Lipid peroxidation was significantly increased in A background strains and in an H-2 dependent manner. The relationship between Ah responsiveness and reported longevities of these nine strains is discussed.

Differential effect of β-naphthoflavone on promutagen activation capabilities of S9 from rat and mouse livers

Differential effect of β-naphthoflavone on promutagen activation capabilities of S9 from rat and mouse livers

Effect of β-Naphthoflavone on Calf Liver Metabolism of Aflatoxin B1

Eight male Holstein calves were raised to 6 wk of age on whole milk at 8% of body weight and calf starter ration (15% crude protein) free choice. For each of 2 days prior to sacrifice, four of the calves were selected at random and administered the mixed function oxidase inducer β-napththoflavone by bolus in starch carrier at .005% of metabolic body weight. After sacrifice, 10,000×g postmitochondrial supernatant preparations were made from the excised livers and incubated with a 50-μg challenge of aflatoxin B1. Although there was no significant difference in total percent of the challenge metabolized, there was a large increase in aflatoxin M1 production by liver preparations from calves administered the flavone inducer.If the flavone family of inducers could alter the metabolic profile for aflatoxin in the adult lactating bovine, aflatoxin M1 excretion rates could be increased and pathways for production of mutagenic metabolites could be enhanced.

The 1,2-dichloroethylenes: Their metabolism by hepatic cytochrome P-450 in vitro

Cis- and trans-1,1-dichloroethylene bound to the active site of hepatic microsomal cytochrome P-450 with the production of a Type I difference spectrum and stimulated CO-inhibitable hepatic microsomal NADPH oxidation. Incubation of cis- and trans-1,2-dichloroethylene plus hepatic microsomes, NADPH-generating system-EDTA resulted in the production of measurable levels of 2,2-dichloroethanol and dichloroacetaldehyde but not of 2-chloroethanol, chloroacetaldehyde or chloroacetic acid and, also, resulted in decreased levels of hepatic microsomal cytochrome P-450 and heme. In addition, dichloroacetic acid was produced from trans-dichloroethylene under these experimental conditions. The omission of any component of the incubation mixture eliminated the above effects, while the inclusion of SKF-525A, metyrapone or CO: O 2 (80, v/v) diminished these effects. The effects of β-naphthoflavone and phenobarbital pretreatment on the values of K s , ΔA max , K m and V mam for the binding and metabolism of the 1,2-dichloroethylenes are reported. The binding and metabolism of the 1,2-dichloroethylenes and the 1,2-dichloroethylene-mediated inactivation of cytochrome P-450 were enhanced per mg of microsomal protein, but generally not per nmole of cytochrome P-450 by prior induction with β-naphthoflavone or phenobarbital. It is concluded that multiple forms of hepatic microsomal cytochrome P-450 bind and metabolize the 1,2-dichloroethylenes. The role of cytochrome P-450 in the metabolic activation of the dichloroethylenes is considered.

The effect of β-naphthoflavone on rabbit liver protein synthesis, and on the induction of cytochrome P-450 LM 4 mRNA

A single injection of β-naphthoflavone dispersed in corn oil causes significant changes in rabbit liver polysome and polysomal poly(A+)mRNA driven in vitro protein synthesis. The changes occur between 6–18 hr and 30–36 hr after the injection. Our data indicate that the first effect is due to the β-naphthoflavone and the second effect is due to the oil vehicle. In vitro translation of rabbit liver polysomes obtained from treated rabbits followed by specific immunoprecipition and gel electrophoresis, showed that maximal levels of translatable cytochrome P-450 LM 4 occurred 18–24 hr after β-naphthoflavone treatment.