Upregulation Of Cytochrome P450 Research Articles

Alterations in histopathology and stress-associated gene expression induced by infection with Prohemistomum vivax encysted metacercariae in Nile tilapia

Due to limited data on the pathogenicity of Prohemistomum vivax (P. vivax) and its impacts on fish health, this study aimed to determine the morphological, molecular characteristics, pathogenicity, and histopathological alterations in fish infected with P. vivax. Eight hundred (800) Nile tilapia (Oreochromis niloticus) were collected from various farms in Kafr El Sheikh Governorate. The fish were examined for encysted metacercariae (EMC) in different organs. Tissue specimens were collected and underwent histopathological analysis, expression of stress-related genes, and genetic characterization by sequencing of the internal transcribed spacer 2 (ITS2). P. vivax metacercariae were oval to round in shape and were collected from various organs including the muscle, skin, eyes, intestine, liver, kidney, and gills of infected O. niloticus. Sequencing and phylogenetic analysis of the ITS2 region revealed a 507-bp fragment, confirming parasite identity and matching within the same clade as other P. vivax isolates. Infected fish displayed abdominal hydropsy, skin darkening, and emaciation. P. vivax encysted metacercariae were detected during the study period in 620/800 fish, with an overall prevalence of 77.5%. The seasonal prevalence was 95% in summer, 85% in spring, 55% in autumn, and 75% in winter. The intensity of infection was 1–40 cysts per microscopic field. Histopathological examination of muscles revealed parasitic cysts embedded within muscle fibers, causing severe degeneration and necrosis. Upregulation of cytochrome P450 (cpy1a1), heat shock protein 70 (hsp-70), and tumor suppressor p53 (p53) was recorded in both liver and muscle samples of infected tilapia compared to controls. This indicates activation of detoxification, cellular stress, and apoptotic pathways in response to P. vivax infection. There is limited data available on the pathogenicity of P. vivax and its impacts on fish health; thus, this study provides key insights into the morphology, pathogenicity, and histopathological impacts of P. vivax in Nile tilapia.

Bufotenines-loaded liposome exerts anti-inflammatory, analgesic effects and reduce gastrointestinal toxicity through altering lipid and bufotenines metabolism

Bufotenines, a natural component from toad venom, showed great potential for development as a novel anti-inflammation and analgesia agent, but the potential toxicity limited its clinic use. In this paper, bufotenines-loaded liposome was prepared and optimized. Then, the therapeutic effects and drug safety of bufotenines-liposome were investigated against inflammation and pain on animal models, with a focus on gastrointestinal toxicity. Bufotenines and its liposome significantly increased paw withdrawal mechanical threshold (PWMT) in Von Frey test and hot paw withdrawal latency (HPWL) in hot-plate test. Moreover, intestinal absorption in vitro and pathological analysis in vivo showed that total bufotenines-loaded liposome significantly reduced the gastrointestinal irritation through reducing exposure of total bufotenines on intestinal tissue. High-sensitivity lipidomics analysis revealed the effect of total bufotenines-loaded liposome were be related to the down-regulation of inflammatory mediators from cyclooxygenase (COX) and lipoxygenase (LOX), the up-regulation of cytochrome P450 (CYP450), and other pathways, thus regulating lipid metabolism pathway and ultimately reducing gastrointestinal irritation. This study shows that liposome-loaded bufotenines has anti-inflammatory, analgesic effects and achieves toxicity reduction. These results provide systematic evidences for efficacy and safety of toad venom active ingredients.

Chronic near lifetime toluene exposure in rodents does not replicate solvent abuse leukoencephalopathy in humans

Toluene is an organic solvent used in industry and as a substance of abuse. The latter situation may be associated with a leukoencephalopathy characterized by white matter atrophy, multifocal myelin loss, and macrophages that contain birefringent granular inclusions. To determine if rodents can develop the same white matter damage, we studied archived rodent brain samples from three near-lifetime toluene carcinogenicity experiments. Rats and mice were exposed to toluene via an inhalation chamber at 1200 ppm for 6.5 h daily, 5 days per week, for 103 weeks. Rats were exposed to toluene via oral gavage of 800 mg/kg, 4 days per week, for 104 weeks. In gavage-exposed brains, immunohistochemical staining was used to detect reactive astroglial and microglial changes, neuron populations, and cytochrome P450 upregulation. None of the white matter changes reported in human toluene abuse were identified in the rat or mouse brains. In a blinded analysis, a mild widespread increase in reactive microglia was detected in female rats that received toluene by gavage at 800 mg/kg. However, no significant differences were detected in neurons or astrocytes. Potential reasons for the absence of changes are discussed. We conclude that rodent studies designed to study carcinogenicity of toluene might not adequately model abuse exposure.

Role of the brain in adaptation of the common and golden carps to adverse factors of chemical origin

Using an indirect solid-phase immunoassay (ELISA test), cytochrome P-450, serotoninmodulated anticonsolidation protein (SMAP) and 70-kDa heat shock protein (HSP70) levels were analyzed in the brain and liver of the common carp Cyprinus carpio L. and golden carp Carassius auratus caught in reservoirs of the Arpachay river (clean zone) and Araks river (polluted zone) in Nakhchivan Autonomous Republic of Azerbaijan. Upregulation of cytochrome P-450 (p < 0.05) and SMAP (p < 0.01) in the common carp brain and downregulation of cytochrome P-450 (p < 0.05) and HSP70 (p < 0.05) in the golden carp liver were detected in fish caught in the Araks river reservoir as compared to levels of the same substances in fish from the Arpachay river reservoir. It is concluded that the brain plays a regulatory role in adaptation of animals to adverse environmental conditions and in protection of tissues from negative effects of pollutants.

Cigarette smoke but not electronic cigarette aerosol activates a stress response in human coronary artery endothelial cells in culture

BackgroundIt is generally acknowledged that e-cigarettes are unlikely to be as harmful as conventional cigarettes, but there is little data that quantifies their relative harms. We investigated the biological response to e-cigarette aerosol exposure (versus conventional cigarette smoke exposure) at the cellular level, by exposing human coronary artery endothelial cells (HCAEC) to aqueous filtered extracts of e-cigarette aerosol or cigarette smoke and looking at gene expression changes consistent with a stress response. This included genes controlled by the oxidant-stress sensing transcription factor NFR2 (NFE2L2), and cytochrome P450 family members. MethodsCigarette smoke extract (CSE) was created using mainstream smoke from a single cigarette drawn through 10ml of endothelial cell growth media MV2. Electronic cigarette aerosol extract (eCAE) was created using the same apparatus, using a constant power output of 10.8w (4.2V) and 18mg/ml nicotine solution. eCAE was generated using 5 cycles of 5s heat with at least 10s in between each puff to allow the coil to cool, air being drawn through the device at 70ml/minute. ResultsHCAEC responded to the noxious components in CSE, resulting in activation of NRF2 and upregulation of cytochrome p450. However, eCAE did not induce NRF2 nuclear localisation, upregulation of NRF2-activated genes, or the upregulation of cytochrome p450. ConclusionsThe use of e-cigarettes as a substitute for conventional cigarettes is likely to reduce immediate tobacco-related harm, at least with respect to cardiovascular harms.

The Cytochrome P450 gene CYP6P12 confers pyrethroid resistance in kdr-free Malaysian populations of the dengue vector Aedes albopictus.

Control of Aedes albopictus, major dengue and chikungunya vector, is threatened by growing cases of insecticide resistance. The mechanisms driving this resistance remain poorly characterised. This study investigated the molecular basis of insecticide resistance in Malaysian populations of Ae. albopictus. Microarray-based transcription profiling revealed that metabolic resistance (cytochrome P450 up-regulation) and possibly a reduced penetration mechanism (consistent over-expression of cuticular protein genes) were associated with pyrethroid resistance. CYP6P12 over-expression was strongly associated with pyrethroid resistance whereas CYP6N3 was rather consistently over-expressed across carbamate and DDT resistant populations. Other detoxification genes also up-regulated in permethrin resistant mosquitoes included a glucuronosyltransferase (AAEL014279-RA) and the glutathione-S transferases GSTS1 and GSTT3. Functional analyses further supported that CYP6P12 contributes to pyrethroid resistance in Ae. albopictus as transgenic expression of CYP6P12 in Drosophila was sufficient to confer pyrethroid resistance in these flies. Furthermore, molecular docking simulations predicted CYP6P12 possessing enzymatic activity towards pyrethroids. Patterns of polymorphism suggested early sign of selection acting on CYP6P12 but not on CYP6N3. The major role played by P450 in the absence of kdr mutations suggests that addition of the synergist PBO to pyrethroids could improve the efficacy of this insecticide class and overcome resistance in field populations of Ae. albopictus.

Tryptophan Catabolites Directly Modulate the Immunosuppressive Effects of MSCs Via Activation of the Endogenous Aryl Hydrocarbon Receptor

It has been demonstrated that the catalytic degradation of tryptophan (Trp) by indoleamine 2,3-dioxygenase (IDO) is a crucial determinant of the immunomodulatory effects of human mesenchymal stromal cells (MSCs). The degradation of Trp has been shown as a mechanism to deprive actively dividing T cells of an amino acid essential for proliferation. However, the deprivation model has come into question by studies showing IDO-catalyzed Trp metabolites exert bioactivity via the aryl hydrocarbon receptor (AHR). AHR is a cytosolic protein expressed by a wide array of lymphomyeloid cells, that upon activation translocates to the nucleus to initiate transcription at xenobiotic response elements (XREs). Molecular activation of AHR is often shown by the upregulation of cytochrome p450 (Cyp) enzymes, Cyp1a1 and Cyp1b1. Since MSCs are known to constitutively express AHR at a basal state, we propose that the catalytic activity of IDO and signal transduction via AHR is linked, as part of an intracellular signaling pathway which deploys MSC suppressive properties. To test this hypothesis, we treated MSCs with aromatic hydrocarbons known to activate AHR: TCDD or FICZ (10 nM), ± IFN-γ (50 ng/mL). In confirmation of previous reports, we showed that human MSCs express AHR at a basal state, and that expression does not change during 48h of treatment with IFN-γ (50 ng/mL); whereas under the same conditions IFN-γ induced expression of IDO RNA transcripts by 104 to 105-fold. Upon treatment of resting MSCs (IDO negative) with the AHR ligand TCDD or FICZ (both at 10nM), we observed 50- to 100-fold induction of Cyp1a1 or Cyp1b1 mRNA, reflecting AHR-induced gene activation. We next examined the response of MSCs after 48h treatment with 1-methyl-tryptophan (1-MT), Trp, kynurenine (Kyn) or kynurenic acid (KynAc), the latter two molecules being the first and second IDO-catalyzed Trp metabolites. Each molecule was tested at final concentrations of 50, 500, and 1000 µM, ± IFN-γ (50 ng/mL). We observed that 1-MT and KynAc both induced Cyp1a1 and Cyp1b1 expression by 20- to 60-fold, with the greatest inductions being seen in cells co-treated with IFN-γ. These changes were observed to occur with the same kinetics as TCDD and FICZ treatments. Neither Trp nor Kyn alone induced Cyp1a1/Cyp1b1 expression. These data support the theory that IDO-driven catabolism of Trp generates endogenous ligand(s) such as KynAc which can directly activate AHR and its signaling pathway. To determine whether Trp catabolites can directly augment the immune suppressive function of MSCs, we performed T cell suppression assays in the presence of MSCs. Whereas activated T cells are unaffected by Kyn or KynAc, we observed that KynAc significantly enhanced the suppressive capacity of MSCs with proliferating T-cells. Interestingly, exposure to Trp catabolites did not alter MSC expression of IDO, HLA-I, HLA-DR or PD-L1 suggesting that AHR activation leads to PD-L1 and IDO-independent suppressive pathways. In conclusion, we demonstrate that MSCs express the AHR in a resting state, and treatment with the prototypical aryl hydrocarbons TCDD or FICZ deploys an AHR-driven molecular genetic response. Further, we show that 1-MT, as well as IDO-catalyzed breakdown products of Trp, can deploy the same AHR-driven molecular signature. Treatment with these aromatic hydrocarbons modulates MSC functionality, leading to altered immunosuppression. Our finding that aryl hydrocarbon treatment of MSCs initiates the AHR response suggests that endogenous AHR signals arising from IDO catalysis may play a role in MSC immunomodulation. This observation supports an entirely novel paradigm for intracellular signaling arising from catabolic conversion of Trp to AHR ligands and induction of immunomodulatory properties. DisclosuresNo relevant conflicts of interest to declare.

Quercetin Induces Hepatic Lipid Omega-Oxidation and Lowers Serum Lipid Levels in Mice

Elevated circulating lipid levels are known risk factors for cardiovascular diseases (CVD). In order to examine the effects of quercetin on lipid metabolism, mice received a mild-high-fat diet without (control) or with supplementation of 0.33% (w/w) quercetin for 12 weeks. Gas chromatography and 1H nuclear magnetic resonance were used to quantitatively measure serum lipid profiles. Whole genome microarray analysis of liver tissue was used to identify possible mechanisms underlying altered circulating lipid levels. Body weight, energy intake and hepatic lipid accumulation did not differ significantly between the quercetin and the control group. In serum of quercetin-fed mice, triglycerides (TG) were decreased with 14% (p<0.001) and total poly unsaturated fatty acids (PUFA) were increased with 13% (p<0.01). Palmitic acid, oleic acid, and linoleic acid were all decreased by 9–15% (p<0.05) in quercetin-fed mice. Both palmitic acid and oleic acid can be oxidized by omega (ω)-oxidation. Gene expression profiling showed that quercetin increased hepatic lipid metabolism, especially ω-oxidation. At the gene level, this was reflected by the up-regulation of cytochrome P450 (Cyp) 4a10, Cyp4a14, Cyp4a31 and Acyl-CoA thioesterase 3 (Acot3). Two relevant regulators, cytochrome P450 oxidoreductase (Por, rate limiting for cytochrome P450s) and the transcription factor constitutive androstane receptor (Car; official symbol Nr1i3) were also up-regulated in the quercetin-fed mice. We conclude that quercetin intake increased hepatic lipid ω-oxidation and lowered corresponding circulating lipid levels, which may contribute to potential beneficial effects on CVD.

Upregulation of cytochrome P450 2J3/11,12-epoxyeicosatrienoic acid inhibits apoptosis in neonatal rat cardiomyocytes by a caspase-dependent pathway

Short, nonlethal ischemic episodes administered to hearts directly after ischemic events (ischemic postconditioning, IPost) have an advantage over ischemic preconditioning (IPC). The endogenous cytochrome P450 2J3/11,12-epoxyeicosatrienoic acid (CYP2J3/11,12-EET) is upregulated by IPost, but not IPC, in the rat heart. The CYP epoxygenase inhibitor N-methylsulphonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH) reduces the cardioprotective effects of IPost, but not IPC. We proposed that upregulation of CYP2J3/11,12-EET during IPost induces cardioprotection by inhibiting cardiomyocyte apoptosis and that multiple apoptotic signals, including changes in mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) opening, mitochondrial cytochrome c leakage, caspase-3 levels, and levels of protective kinases such as Bcl-2 and Bax, are involved in the process. Neonatal rat cardiomyocytes underwent 3-h hypoxia followed by 2-, 5-, or 6-h reoxygenation (H/R) or three cycles of 5-min reoxygenation followed by 5-min hypoxia before 90-min reoxygenation (HPost); or were transfected with pcDNA3.1-CYP2J3 for 48h before H/R; or were treated with MS-PPOH for 10min before HPost. For HPost alone, pcDNA3.1-CYP2J3 transfection attenuated cardiomyocyte apoptosis to 68.4% (p<0.05) of that with H/R. pcDNA3.1-CYP2J3 transfection significantly decreased MMP and inhibited mPTP opening induced by H/R, reduced mitochondrial cytochrome c leakage, cleaved caspase-3 protein expression, and increased the ratio of Bcl-2 to Bax expression. MS-PPOH abolished this effect. Therefore, upregulation of CYP2J3/11,12-EET during HPost is involved in cardioprotection by inhibiting apoptosis via a caspase-dependent pathway, and the apoptosis-suppressive effect may have important clinical implications during HPost.

Multidrug resistance protein (MRP) 4 attenuates benzo[ a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells

Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[ a]pyrene (B[ a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) or (−)-benzo[ a]pyrene-7,8-dihydro-7,8-diol (B[ a]P-7,8-dihydrodiol), the proximate carcinogen of B[ a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene-2′-deoxyguanosine ((+)- anti-trans-B[ a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)- anti-trans-B[ a]PDE-dGuo formation in cells treated with (−)-B[ a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)- anti-trans-B[ a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (−)-B[ a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen.

RNAi-Based Screening Identifies Kinases Interfering with Dioxin-Mediated Up-Regulation of CYP1A1 Activity

BackgroundThe aryl hydrocarbon receptor (AhR) is a transcription factor activated by several environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and involved in carcinogenesis and various physiological processes, including immune response and endocrine functions. Characterization of kinases-related AhR transduction pathway remains an important purpose.ResultsWe performed a kinome-wide siRNA screen in human mammary MCF-7 cells to identify non redundant protein kinases implicated in the up-regulation of cytochrome P-450 (CYP) 1A1 activity, an AhR referent target, in response to TCDD exposure. To this aim, we monitored CYP1A1-related ethoxyresorufin-O-deethylase (EROD) activity and quantified cell density. This normalization was crucial since it allowed us to focus only on siRNA affecting EROD activity and discard siRNA affecting cell density. Analyses of the cell density data allowed us to identify several hits already well-characterized as effectors of the cell cycle and original hits. Collectively, these data fully validated the protocol and the siRNA library. Next, 22 novel candidates were identified as kinases potentially implicated in the up-regulation of CYP1A1 in response to TCDD, without alteration of cell survival or cell proliferation. The siRNA library screen gave a limited number of hits (approximately 3%). Interestingly, four of them are able to bind calmodulin among which the IP3 kinase A (ITPKA) and pregnancy up-regulated non-ubiquitously expressed CaM kinase (PNCK, also named CaMKIβ). Remarkably, for both proteins, their kinase activity depends on the calmodulin binding. Involvement of ITPKA and PNCK in TCDD-mediated CYP1A1 up-regulation was further validated by screening-independent expression knock-down. PNCK was finally shown to regulate activation of CaMKIα, a CaMKI isoform previously reported to interplay with the AhR pathway.ConclusionsThese data fully support a role for both IP3-related kinase and CaMK isoforms in the AhR signaling cascade. More generally, this study also highlights the interest of large scale loss-of-function screens for characterizing the molecular mechanism of action of environmental contaminants.

Up-regulation of cytochrome P450 and phase II enzyme systems in rat precision-cut rat lung slices by the intact glucosinolates, glucoraphanin and glucoerucin

It is believed that the chemopreventive activity of cruciferous vegetables in the lung and other tissues is exclusively the result of exposure to degradation products of glucosinolates, such as the isothiocyanates, and that the parent glucosinolates make no contribution. In the present study, evidence is presented for the first time that, in rat lung, the intact glucosinolates, glucoraphanin and glucoerucin, can modulate carcinogen-metabolising enzyme systems. The glucosinolates were isolated from cruciferous vegetables and incubated (1–25μM) with precision-cut rat lung slices for 24h. Both glucosinolates, at concentrations as low as 1μM, up-regulated the O-deethylation of ethoxyresorufin and the apoprotein levels of CYP1A1 and CYP1B1; supplementation of the incubation medium with myrosinase, the enzyme that converts glucosinolates to their corresponding isothiocyanates, abolished the rise in ethoxyresorufin O-deethylase activity. In contrast, neither glucosinolate, at the concentrations studied, influenced quinone reductase activity in the lung slices, but addition of myrosinase to the glucosinolate incubations led to a marked rise in activity. Glutathione S-transferase activity, monitored using 1-chloro-2,4-dinitrobenzene as the accepting substrate, was elevated in lung slices exposed to glucoraphanin. GSTα protein levels were increased by glucoraphanin and, to a much lesser extent, glucoerucin. It may be concluded that intact glucosinolates can modulate the activity of pulmonary carcinogen-metabolising enzyme systems, and can thus contribute to the documented chemopreventive activity of cruciferous vegetables in the lung.

Role of transcription factors in estrogen‐dependent upregulation of CYP 2C family in arteries of eNOS deficient mice

Upregulation of cytochrome P450 (CYP) 2C29/38/40 is responsible for the EET‐mediated flow‐dependent dilation in microvessels of female mice, as a function of NO deficiency. In the present studies, we provide evidence of the correlation between transcription factors (PPAR‐γ, PPAR‐δ and RXR‐γ) and CYP 2C29/38/40 in isolated arterioles of female eNOS‐KO mice by microarray (SuperArray, OMM‐401). Five single arterioles isolated from mesentery of both genders of eNOS‐KO and WT mice were pooled and snap frozen in liquid nitrogen; ∼10 μg of total RNA were isolated, and a total of 10 arrays from 2 WT and 3 eNOS‐KO mice of each gender were performed. Microarray results indicate that associated with a specific upregulation of CYP 2C29/38/40, expression of genes for PPAR‐γ, PPAR‐δ and RXR‐γ, the nuclear receptors that function as gene regulators via specific DNA binding, was also increased (P&lt;0.01, 0.05 and 0.03, respectively) in female eNOS‐KO compared to male eNOS‐KO vessels. These significant differences were validated by quantitative real‐time PCR and Western blot analysis. The increase in gene expression for the three transcription factors was only observed in vessels of female, but not in those of male eNOS‐KO mice, and without significant differences between vessels of WT male and WT female mice, a pattern that is similar to the upregulation of CYP 2C/29/38/40. Thus, the microarray analysis screened the global expression of genes for the transcription factors and revealed the specificity of estrogen and NO deficiency in the control of the transcription factors which may play a role in the regulation of CYP 2C expression (Supported by NIH HL‐43023, HL‐68813 and HL‐070653).

Growth kinetics in MCF-7 cells modulate benzo[a]pyrene-induced CYP1A1 up-regulation

Pro-carcinogens, such as benzo[a]pyrene (B[a]P), that are exogenous ligands of the aromatic hydrocarbon receptor may influence the susceptibility of target-cell populations through the up-regulation of cytochrome P450 (CYP) mixed function oxidases. We examined whether the growth kinetics of MCF-7 cells might determine the level of up-regulation of CYP1A1, CYP1A2 or CYP1B1 by B[a]P, and whether this could then influence subsequent levels of DNA damage. Cell cultures manipulated to be G(0)/G(1)-phase concentrated, S-phase concentrated or G(2)/M-phase concentrated were treated with B[a]P and the expression levels of CYP1A1, CYP1A2, CYP1B1, cyclin-dependent kinase inhibitor 1A [CDKN1A (P21(WAF1/CIP1))], B-cell leukaemia/lymphoma-2 (BCL-2), and Bcl-2-associated X levels were determined. Levels of DNA damage were measured as DNA single-strand breaks (SSBs) by the alkaline single-cell gel electrophoresis (comet) assay or as DNA adducts by (32)P-postlabelling analysis. B[a]P-induced up-regulation of CYP1A1 was >100-fold in S-phase-concentrated cells, but in G(0)/G(1)-phase- or G(2)/M-phase-concentrated cultures up-regulation occurred to a significantly lower extent. Consistent with this, B[a]P-treated S-phase-concentrated cultures exhibited markedly up-regulated P21(WAF1/CIP1), higher levels of dose-related increases in DNA SSBs, and increased DNA adduct levels presumably as a result of CYP1A1-mediated activation of B[a]P to B[a]P-diol-epoxide compared with the cultures enriched for the other cell cycle phases. Growth kinetics in vitro may be an important predeterminant of susceptibility to an exogenous pro-carcinogen in short-term test systems and these findings have important implications when extrapolating such results to a particular target-cell population in vivo.

A novel synthetic analogue of a constituent of Isodon excisus inhibits transcription of CYP1A1, -1A2 and -1B1 by preventing activation of the aryl hydrocarbon receptor

We investigated the effect of a novel synthetic analogue of a constituent from the Chinese medicinal herb Isodon excisus, 3-(3-methoxy-phenyl)-N-(3, 4, 5-trimethoxy-phenyl)-acrylamide (compound 343), on the carcinogen activation pathway mediated by the aryl hydrocarbon receptor (AhR) in human hepatoma HepG2 cells. We found that compound 343 inhibited the upregulation of cytochrome P-450 (CYP) enzyme activity in cells treated with the AhR ligands and potent carcinogens, dimethylbenz[a]anthracene (DMBA) or 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Compound 343 also inhibited the DMBA- or TCDD-induced increase in CYP1A1, -1A2 and -1B1 mRNA levels. Carcinogen-induced transcription of CYP genes was also suppressed by compound 343, as measured by a reporter gene controlled by the xenobiotic-responsive element (XRE). This was confirmed by measuring the amount of carcinogen-induced CYP1A1 heterogeneous nuclear RNA. Compound 343 blocked the DMBA- or TCDD-induced activation of the AhR DNA-binding capacity for the XRE, as measured by a chromatin immunoprecipitation assay. Compound 343 also inhibited CYP enzyme activity in microsomes isolated from DMBA- or TCDD-treated cells, as well as the activity of recombinant CYP1A1, -1A2 and -1B1, indicating that compound 343 directly inhibits CYP enzymes. These results indicate that compound 343 is both a potent inhibitor of carcinogen-induced CYP enzyme expression, as well as a direct inhibitor of CYP enzymes.

Attenuation of Hyperoxic Lung Injury by the CYP1A Inducer β–Naphthoflavone

Supplemental oxygen, frequently used in premature infants, has been implicated in the development of bronchopulmonary dysplasia (BPD). While the mechanisms of oxygen-induced lung injury are not known, reactive oxygen species (ROS) are most likely involved in the process. Here, we tested the hypothesis that upregulation of cytochrome P450 (CYP) 1A isoforms in lung and liver may lead to protection against hyperoxic lung injury. Adult male Sprague-Dawley rats were pretreated with the CYP1A inducer beta-naphthoflavone (beta-NF) (80 mg/kg/day), once daily for 4 days, followed by exposure to hyperoxic environment (O2 > 95%) or room air (normoxia) for 60 h. Pleural effusions were measured as estimates of lung injury. Activities of hepatic and pulmonary CYP1A1 were determined by measurement of ethoxyresorufin O-deethylation (EROD) activity. Northern hybridization and Western blot analysis of lung and liver were performed to assess mRNA and protein levels, respectively. Our results showed that beta-NF-treated animals, which displayed the highest pulmonary and hepatic induction in EROD activity (10-fold and 8-fold increase over corn oil (CO) controls, respectively), offered the most protective effect against hyperoxic lung injury, p < 0.05. Northern and Western blot analysis correlated well with enzyme activities. Our results showed an inverse correlation between pulmonary and hepatic CYP1A expression and the extent of lung injury, which supports the hypothesis that CYP1A enzyme plays a protective role against oxygen-mediated tissue damage.

Pretranslational upregulation of microsomal CYP4A in rat liver by intake of a high-sucrose, lipid-devoid diet containing orotic acid

In rodents, high-fat diets promote hepatic lipid accumulation in rodents, activation of peroxisome proliferator activated receptor-α (PPARα) and upregulation of cytochrome P450 (CYP) 4A gene expression. Lipid-devoid diets containing sucrose and orotic acid (S/OA-diet) also cause lipid infiltration by stimulating intrahepatic lipid synthesis and preventing lipoprotein transport through the Golgi apparatus. This study evaluated the impact of the lipid-deficient S/OA-diet on CYP4A expression and PPARα activation in rodent liver. CYP4A protein and laurate ω-hydroxylation activity were increased in rat liver after S/OA-feeding for 21 days. CYP4A1 and CYP4A2 mRNAs were induced to 2.1- and 2.6-fold of control, but mRNAs corresponding to CYP4A3 and the peroxisomal acyl-CoA oxidase (AOX) were unchanged. Coadministration of clofibric acid and the S/OA-diet prevented hepatic lipid accumulation and upregulated CYP4A protein to levels comparable with clofibric acid alone (five-fold of control). Clofibric acid, alone and in combination with the S/OA-diet, upregulated CYP4A1-3 and AOX mRNAs. Hepatic PPARα protein was decreased by the S/OA-diet but was increased to 5.7-fold of control by clofibric acid; retinoid X-receptor-α (RXRα) protein was decreased to 26–41% of control by all treatments. In further studies, administration of the S/OA-diet to control and PPARα-null mice promoted hepatic lipid deposition; microsomal CYP4A protein was induced in wild-type but not PPARα-null mice. These findings implicate PPARα in the induction of CYP4A in rodent liver by the lipid-devoid S/OA-diet. Decreased availability of hepatic PPARα and RXRα after intake of the diet may contribute to the selective upregulation of hepatic CYP4A1 and CYP4A2 in this model.