Hepatic Biotransformation Enzymes Research Articles

Interaction of Bisphenol A and Its Analogs with Estrogen and Androgen Receptor from Atlantic Cod (Gadus morhua).

The widespread use of bisphenol A (BPA) in polycarbonate plastics and epoxy resins has made it a prevalent environmental pollutant in aquatic ecosystems. BPA poses a significant threat to marine and freshwater wildlife due to its documented endocrine-disrupting effects on various species. Manufacturers are increasingly turning to other bisphenol compounds as supposedly safer alternatives. In this study, we employed in vitro reporter gene assays and ex vivo precision-cut liver slices from Atlantic cod (Gadus morhua) to investigate whether BPA and 11 BPA analogs exhibit estrogenic, antiestrogenic, androgenic, or antiandrogenic effects by influencing estrogen or androgen receptor signaling pathways. Most bisphenols, including BPA, displayed estrogenic properties by activating the Atlantic cod estrogen receptor alpha (gmEra). BPB, BPE, and BPF exhibited efficacy similar to or higher than that of BPA, with BPB and BPAF being more potent agonists. Additionally, some bisphenols, like BPG, induced estrogenic effects in ex vivo liver slices despite not activating gmEra in vitro, suggesting structural modifications by hepatic biotransformation enzymes. While only BPC2 and BPAF activated the Atlantic cod androgen receptor alpha (gmAra), several bisphenols exhibited antiandrogenic effects by inhibiting gmAra activity. This study underscores the endocrine-disrupting impact of bisphenols on aquatic organisms, emphasizing that substitutes for BPA may pose equal or greater risks to both the environment and human health.

Effects of Echinococcus multilocularis metacestodes infection and drug treatment on the activities of biotransformation enzymes in mouse liver.

The changes of biotransformation enzymes will substantially affect the host's ability to metabolize drugs and other xenobiotic compounds. In order to further elucidate this process and promote the development in treatment of echinococcosis, we investigated the effects of Echinococcus multilocularis infection and drug treatment on biotransformation enzymes in mouse liver. In microsomal and cytosolic fractions, from the six activities assayed, significant decrease of glutathione S-transferases (GST) activity and significant increase of 7-pentoxyresorufin (PROD) and NADPH-cytochrome P450 reductase (CPR) activity were observed in the mice infected withE. multilocularismetacestodes. In addition, after six weeks treatment of albendazole inE. multilocularisinfected mice, significant decreased GST activity and significant increase of 7- ethoxyresorufin (EROD), PROD, and particularly 3-fold higher 7-methoxyresorufin (MROD) activity were observed. The 3-bromopyruvate treated mice only exhibited significantly lower GST activity. Our results demonstrate thatE. multilocularismetacestodes infection can affect the activities of main hepatic biotransformation enzymes and such alterations of activity may further affect the hepatic biotransformation of xenobiotics. Moreover, albendazole and 3-bromopyruvate, the promising potential drug againstEchinococcus, affected different hepatic biotransformation enzymes and may affect their metabolism. The findings will help to develop rational treatments with less side effects and promote the development of more efficient treatments againstE. multilocularis.

Responses of hepatic biotransformation and antioxidant enzymes in Japanese medaka (Oryzias latipes) exposed to humic acid.

Humic substances, a major component of natural organic matter in surface waters, can induce biotransformation enzyme activities and influence antioxidant defense in fish. The study aimed to provide a molecular basis for the stress responses, the synthesis of biotransformation, and antioxidant enzymes in particular. Adult medaka fish (Hd-rR strain) were exposed to environmentally relevant concentrations of humic acid for 96h. The actual humic acid concentrations in water were determined photometrically and expressed as organic carbon concentrations. Liquid chromatography with tandem mass spectrometry was used for protein profile analysis of medaka liver samples. The relative amount of isozymes was determined using the label-free quantification approach. Hepatic biotransformation enzyme activities were measured as well. Thus, ethoxyresorufin-O-deethylase activity showed a pronounced induction at the highest tested concentration (9.4mg C/L). Various biotransformation and antioxidant isozymes responded to humic acid differently, reflecting a balanced interplay of proteins that ensures the metabolism of humic acid in fish liver. Some isozymes were not affected by humic acid. The study provides new insight into the molecular mechanisms of the fish stress response to the humic acid-related challenge.

Mono-n-Butyl Phthalate Distributes to the Mouse Ovary and Liver and Alters the Expression of Phthalate-Metabolizing Enzymes in Both Tissues.

Humans are exposed to phthalates daily via items such as personal care products and medications. Reproductive toxicity has been documented in mice exposed to di-n-butyl phthalate (DBP); however, quantitative evidence of its metabolite, mono-n-butyl phthalate (MBP), reaching the mouse ovary and its effects on hepatic and ovarian biotransformation enzymes in treated mice is still lacking. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) was employed to quantify MBP levels in liver, serum, and ovary from mice treated with a single or repeated exposure to the parent compound, DBP. Adult CD-1 females were pipet fed once or for 10 days with vehicle (tocopherol-stripped corn oil) or DBP at 1, 10, and 1000 mg/kg/day. Tissues and serum were collected at 2, 6, 12, and 24 h after the single or final dose and subjected to LC-MS/MS. Ovaries and livers were processed for qPCR analysis of selected phthalate-associated biotransformation enzymes. Regardless of duration of exposure (single vs repeated), MBP was detected in the tissues of DBP-treated mice. In single dose mice, MBP levels peaked at ≤6 h and fell close to background levels by 24 h post-exposure. Following the last repeated dose, MBP levels peaked at ≤2 h and fell to background levels by 12 h. Hepatic and ovarian expression of Lpl, Aldh1a1, Adh1, Ugt1a6a, and Cyp1b1 were altered in DBP-treated mice in a time- and dose-specific manner. These findings confirm that MBP reaches the mouse liver and ovary after oral exposure to DBP and influences the expression of hepatic and ovarian phthalate-associated biotransformation enzymes.

Accumulation of human pharmaceuticals and activity of biotransformation enzymes in fish from two areas of the lower Rio de la Plata Basin

The accumulation of four human pharmaceuticals active compounds (HPhAC) in the muscle of four fish species of the Rio de la Plata Basin were assessed regarding the compound logP and pKa, and fish trophic levels. For Prochilodus lineatus, accumulation was compared to hepatic biotransformation enzymes and fish from two sampling areas with different urbanization degree. Species were the detritivore Prochilodus lineatus, the omnivores Megaleporinus obtusidens and Pimelodus maculatus, and the piscivorous Salminus brasiliensis. Sampling areas were the Inner Rio de la Plata Estuary (RLP), in front of the Buenos Aires Metropolitan Area, and at the Lower Uruguay River (URU), a relatively unpopulated area. Carbamazepine, atenolol, enalapril, and sildenafil concentrations in fish muscle were analyzed by HPLC-MS. EROD, BROD, and GST activities were measured in P. lineatus liver. Average (maximum) concentrations and detection frequency were: Atenolol 24.4 (69.4) μg kg−1, 60%; carbamazepine 5.5 (45.8) μg kg−1, 19%; enalapril 7.0 (56.9) μg kg−1, 28%; sildenafil 17.1 (71.6) μg kg−1, 56%. Enalapril and sildenafil detection in fish was first-time reported. Atenolol and carbamazepine concentrations were positively correlated. No correlation was observed between HPhAC accumulation and logP or pKa. A potential biomagnification trend was observed for atenolol, showing higher accumulation in S. brasiliensis. HPhACs accumulation was higher for P. lineatus collected at URU, but GST and BROD were significantly higher for individuals sampled at RLP and were positively correlated. A significant negative correlation was observed for enalapril and BROD. The study contributes to the knowledge of the accumulation of HPhACs in fish.

Effect of polychlorinated biphenyls on oxidation stress in the liver of juvenile GIFT, Oreochromis niloticus.

The present study clearly showed that chronic exposure to polychlorinated biphenyls (PCBs) at environmentally relevant concentrations can damage juvenile tilapia livers by modulating antioxidant enzyme activities and gene transcription, which affects toxic bioaccumulation and histological congestion. The results suggest that PCBs caused a decrease in the activity of some hepatic antioxidative and biotransformation enzymes (SOD, CAT, GST, T-GSH, and MDA) in tilapia at 7 days, as well as transcriptional changes (sod, cat, and gst). Except for some antioxidant parameters (T-GSH, GSH/GSSG, T-AOC, and MDA), significant declines and increases occurred at 14 and 21 days, respectively. Most of the antioxidant enzymatic signatures and genotoxicity significantly increased at 14 and 21 days. This study presented evidence that PCBs could result in hepatic toxicity through oxidative stress in the early growth stages of tilapia, and we speculated that oxidative stress plays an important role in embryonic developmental toxicity induced by PCBs.

Hepatic biotransformation and antioxidant enzyme activities in Mediterranean fish from different habitat depths

Marine fish are threatened by anthropogenic chemical discharges. However, knowledge on adverse effects on deep-sea fish or their detoxification capabilities is limited. Herein, we compared the basal activities of selected hepatic detoxification enzymes in several species (Solea solea, Dicentrarchus labrax, Trachyrhynchus scabrus, Mora moro, Cataetix laticeps and Alepocehalus rostratus) collected from the coast, middle and lower slopes of the Blanes Canyon region (Catalan continental margin, NW Mediterranean Sea). The xenobiotic-detoxifying enzymes analysed were the phase-I carboxylesterases (CbEs), and the phase-II conjugation activities uridine diphosphate glucuronyltransferase (UDPGT) and glutathione S-transferase (GST). Moreover, some antioxidant enzyme activities, i.e., catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR), were also included in this comparative study. Because CbE activity is represented by multiple isoforms, the substrates α-naphthyl acetate (αNA) and ρ-nitrophenyl acetate (ρNPA) were used in the enzyme assays, and in vitro inhibition kinetics with dichlorvos were performed to compare interspecific CbE sensitivity.Activity of xenobiotic detoxification enzymes varied among the species, following a trend with habitat depth and body size. Thus, UDPGT and some antioxidant enzyme activities decreased in fish inhabiting lower slopes of deep-sea, whereas UDPGT and αNA-CbE activities were negatively related to fish size. A trend between CbE activities and the IC50 values for dichlorvos suggested S. solea and M. moro as potentially more sensitive to anticholinesterasic pesticides, and T. scabrus as the most resistant one. A principal component analysis considering all enzyme activities clearly identified the species but this grouping was not related to habitat depth or phylogeny. Although these results can be taken as baseline levels of the main xenobiotic detoxification enzymes in Mediterranean fish, further research is needed to evaluate their response to environmental contaminant exposure.

Hepatic effects of repeated oral administration of diclofenac to hepatic cytochrome P450 reductase null (HRN™) and wild-type mice.

Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit normal hepatic and extrahepatic biotransformation enzyme activities when compared to wild-type (WT) mice, but express no functional hepatic cytochrome P450 activities. When incubated in vitro with [(14)C]-diclofenac, liver microsomes from WT mice exhibited extensive biotransformation to oxidative and glucuronide metabolites and covalent binding to proteins was also observed. In contrast, whereas glucuronide conjugates and a quinone-imine metabolite were formed when [(14)C]-diclofenac was incubated with HRN™ mouse liver, only small quantities of P450-derived oxidative metabolites were produced in these samples and covalent binding to proteins was not observed. Livers from vehicle-treated HRN™ mice exhibited enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration and necrosis and inflammatory cell infiltration, which were not present in livers from WT mice. Elevated liver-derived alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities were also observed in plasma from HRN™ mice. When treated orally with diclofenac for 7 days, at 30 mg/kg/day, the severities of the abnormal liver histopathology and plasma liver enzyme findings in HRN™ mice were reduced markedly. Oral diclofenac administration did not alter the liver histopathology or elevate plasma enzyme activities of WT mice. These findings indicate that HRN™ mice are valuable for exploration of the role played by hepatic P450s in drug biotransformation, but poorly suited to investigations of drug-induced liver toxicity. Nevertheless, studies in HRN™ mice could provide novel insights into the role played by inflammation in liver injury and may aid the evaluation of new strategies for its treatment.

Effect of standardized cranberry extract on the activity and expression of selected biotransformation enzymes in rat liver and intestine.

The use of dietary supplements containing cranberry extract is a common way to prevent urinary tract infections. As consumption of these supplements containing a mixture of concentrated anthocyanins and proanthocyanidins has increased, interest in their possible interactions with drug-metabolizing enzymes has grown. In this in vivo study, rats were treated with a standardized cranberry extract (CystiCran®) obtained from Vaccinium macrocarpon in two dosage schemes (14 days, 0.5 mg of proanthocyanidins/kg/day; 1 day, 1.5 mg of proanthocyanidins/kg/day). The aim of this study was to evaluate the effect of anthocyanins and proanthocyanidins contained in this extract on the activity and expression of intestinal and hepatic biotransformation enzymes: cytochrome P450 (CYP1A1, CYP1A2, CYP2B and CYP3A), carbonyl reductase 1 (CBR1), glutathione-S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Administration of cranberry extract led to moderate increases in the activities of hepatic CYP3A (by 34%), CYP1A1 (by 38%), UGT (by 40%), CBR1 (by 17%) and GST (by 13%), while activities of these enzymes in the small intestine were unchanged. No changes in the relative amounts of these proteins were found. Taken together, the interactions of cranberry extract with simultaneously administered drugs seem not to be serious.

Combined effects of Iranian heavy crude oil and bacterial challenge (Streptococcus iniae) on biotransformation and innate immune responses in rockfish (Sebastes schlegeli).

To clarify the effects of spilled crude oil on fish bacterial disease resistance, rockfish (Sebastes schlegeli) were exposed to Iranian Heavy crude oil (IHCO) and Streptomyces iniae in combination. Hepatic biotransformation enzymes (ethoxyresorufin O-de-ethylase, glutathione-S-transferase) and plasma biochemical parameters (glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and glucose) in fish exposed to IHCO were not significantly different from those in unexposed fish. The level of biliary 1-OH-pyrene and cytochrome P4501A mRNA expression increased in a dose-dependent manner with IHCO exposure. The interferon stimulated gene 15, interleukin-1beta and cathepsin L were increased significantly in the liver in IHCO-exposed fish, but not dose-dependently, but the granulocyte colony stimulating factor was not related to IHCO exposure. The percentage mortality in fish following a single exposure to S. iniae was positively correlated with IHCO exposure concentration. We concluded that IHCO exposure exacerbates fish mortality following environmental bacterial infection.

The human constitutive androstane receptor promotes the differentiation and maturation of hepatic-like cells

Expression of the constitutive androstane receptor (CAR, NR1I3) is enriched in the mature mammalian liver and increasingly recognized for its prominent role in regulating a myriad of processes including biotransformation, chemical transport, energy metabolism and lipid homeostasis. Previously, we demonstrated that CAR levels were markedly enhanced during the differentiation of hepatic-like cells derived from hESCs, prompting the hypothesis that CAR contributes a key functional role in directing human hepatogenesis. Here we demonstrate that over-expression of CAR in human embryonic stem cells (ESCs), transduced by a lentiviral vector, accelerates the maturation of hepatic-like cells, with CAR over-expressing cells exhibiting a 2.5-fold increase in albumin secretion by day 20 in culture differentiation, and significantly enhanced levels of mRNA expression of several liver-selective markers, including hepatic transcription factors, plasma proteins, biotransformation enzymes, and metabolic enzymes. CAR over-expressing cells also exhibited enhanced CITCO-inducible CYP3A7 enzymatic activity. Knockdown of CAR via siRNA attenuated the differentiation-dependent expression programs. In contrast, expression levels of the pregnane X receptor (PXR), a nuclear receptor most similar to CAR in primary sequence, were negligible in human fetal liver tissues or in the differentiating hESCs, and stable over-expression of PXR in hepatic-induced hESCs failed to enhance expression of hepatic phenotype markers. Together, these results define a novel role for human CAR in hepatic lineage commitment.

Effect of Caloric Restriction and AMPK Activation on Hepatic Nuclear Receptor, Biotransformation Enzyme, and Transporter Expression in Lean and Obese Mice

Fatty liver alters liver transporter expression. Caloric restriction (CR), the recommended therapy to reverse fatty liver, increases Sirtuin1 deacetylase activity in liver. This study evaluated whether CR and CR mimetics reversed obesity-induced transporter expression in liver and hepatocytes. mRNA and protein expression was determined in adult lean (lean) and leptin-deficient obese (OB) mice fed ad libitum or placed on 40% (kCal) reduced diet. Hepatocytes were isolated from lean and OB mice, treated with AMP Kinase activators, and gene expression was determined. CR decreased Oatp1a1, Oatp1b2, and Abcb11 mRNA expression in lean, but not OB mice. CR increased Abcc2 mRNA OB livers, whereas protein expression increased in both genotypes. CR increased Abcc3 protein expression increased in OB livers. CR did not alter Abcc1, 4 and 5 mRNA expression in lean mice but decreased expression in livers of OB mice. CR increased Abcc4 protein in lean, but not OB mice. CR restriction reversed the expression of some, but not all transporters in livers of OB mice. Overall, these data indicate a potential for CR to restore some hepatic transporter changes in OB mice, but suggest a functional leptin axis is needed for reversal of expression for some transporters.

Toxic effects of dietary exposure to T-2 toxin on intestinal and hepatic biotransformation enzymes and drug transporter systems in broiler chickens

The effects of the mycotoxin T-2 on hepatic and intestinal drug-metabolizing enzymes (cytochrome P450) and drug transporter systems (MDR1 and MRP2) in poultry were investigated during this study. Broiler chickens received either uncontaminated feed, feed contaminated with 68μg/kg or 752μg/kg T-2 toxin. After 3weeks, the animals were euthanized and MDR1, MRP2, CYP1A4, CYP1A5 and CYP3A37 mRNA expression were analyzed using qRT-PCR. Along the entire length of the small intestine no significant differences were observed. In the liver, genes coding for CYP1A4, CYP1A5 and CYP3A37 were significantly down-regulated in the group exposed to 752μg/kg T-2. For CYP1A4, even a contamination level of 68μg/kg T-2 caused a significant decrease in mRNA expression. Expression of MDR1 was not significantly decreased in the liver. In contrast, hepatic MRP2 expression was significantly down-regulated after exposure to 752μg/kg T-2. Hepatic and intestinal microsomes were prepared to test the enzymatic activity of CYP3A. In the ileum and liver CYP3A activity was significantly increased in the group receiving 752μg/kg T-2 compared to the control group. The results of this study show that drug metabolizing enzymes and drug transporter mechanisms can be influenced due to prolonged exposure to relevant doses of T-2.

D-Pinitol prevents rat breast carcinogenesis induced by 7, 12 -Dimethylbenz [a] anthracene through inhibition of Bcl-2 and induction of p53, caspase-3 proteins and modulation of hepatic biotransformation enzymes and antioxidants

Dietary compounds are reported to have the innate ability to interfere several diseases. D-Pinitol is one of the compounds from dietary origin, well documented for its excellent biological activities. We have reported the therapeutic efficiency of the D-Pinitol with reference to lipid peroxidation, antioxidants, drug metabolizing enzymes and expressions of proteins such as p53, Bcl-2, and caspase-3 during DMBA-induced mammary carcinogenesis. D-Pinitol at the dose of 200mg/kg body weight was administered orally to treat the breast cancer-bearing animals for consecutive 45 days. Interestingly, D-Pinitol significantly decreased the lipid peroxidation and increased the antioxidants and modulated the biotransformation enzymes to near normal level when compared to control. The western blotting and RT-PCR analysis also inevitably confirms that D-Pinitol treatment down regulated the expression of Bcl-2 and up regulated the p53 and caspase-3 proteins. The histological analysis of breast and liver tissues were well supported the protective role of D-Pinitol. Thus, the therapeutic property of D-Pinitol might be due to its strong antioxidant and remarkable induction in phase II and significant modulation in phase I drug metabolizing enzymes and prominent influence in the proteins of apoptotic, anti-apoptotic and tumor suppressors which ultimately end up with the consideration of D-Pinitol in the treatment of genotoxin mediated carcinogenesis.

Therapeutic effect of hesperidin with reference to biotransformation, lysosomal and mitochondrial TCA cycle enzymes against 7,12-dimethylbenz(a)anthracene-induced experimental mammary cellular carcinoma

The present study was aimed to investigate the therapeutic effect of hesperidin with reference to hepatic biotransformation, lysosomal and mitochondrial TCA cycle enzymes against 7,12-dimethylbenz(a)anthracene (DMBA)-induced experimental breast cancer. DMBA is a major class of chemical carcinogen present in the environment and it may increase breast cancer risk. In the present investigation, 7,12-DMBA was used as a carcinogen to induce breast cancer in rats. Hesperidin, a natural flavanoglycoside has demonstrated a wide variety of biological activities which make it a good candidate for the treatment of many oxidative stress-mediated diseases. In this study the hepatic biotransformation (Phase I, Phase II) enzymes were analyzed both in breast and liver. The lysosomal enzymes were analyzed in breast tissues and the key mitochondrial TCA cycle enzymes were analyzed in liver. The results revealed that treatment with hesperidin (30 mg/kg body weight) significantly restored all the altered biochemical parameters. The ultra structural analysis of breast and histology of liver tissues inevitably supports the biochemical alterations and this was attributed due to the interaction of flavonoid hesperidin through the induction or inhibition of metabolism and also the modulating property in the lysosomal and TCA cycle compartments.

Hepatic biotransformation genes and enzymes and PAH metabolites in bile of common sole (Solea solea, Linnaeus, 1758) from an oil-contaminated site in the Mediterranean Sea: A field study

The aim of the present field study was to evaluate the response of hepatic biotransformation genes and enzymes of the common sole collected from an area characterized by the presence of an oil-refinery. Based on ∑PAHs levels detected in sediments, three sites were identified: an high-impact site in front of the refinery, a moderate impact site and a reference site at increasing distances from the refinery. Transcription of cyp1a, udpgt and gst genes and related enzyme activities, such as EROD, BROD, MROD, B(a)PMO, UDPGT and GST, were assessed in sole liver. PAHs bile metabolites were measured. The link between phases I and II is discussed with regard to levels of PAHs measured in sediments and fillets. Results provide sequencing data on biotransformation genes essential for further studies on transcriptional responses in common sole and confirm phase I enzyme activities as useful tools for future biomonitoring studies in marine coastal areas.

CYP1A2*1F and GSTM1 Alleles Are Associated with Susceptibility to Porphyria Cutanea Tarda

Porphyria cutanea tarda (PCT) is a cutaneous porphyria with sporadic (type 1) and familial (type 2) subtypes, both resulting from decreased hepatic uroporphyrinogen decarboxylase (UROD) activity. Environmental and genetic factors are involved in the development of PCT, and genetic variants in the cytochrome P450 (CYP ) genes, CYP1A1 and CYP1A2, have been implicated. We investigated the association between PCT and variants in CYP1A1, CYP1A2 and CYP2E1, and the glutathione-S-transferase (GST ) genes, GSTM1 and GSTT1. PCT diagnosis was based on urinary or plasma porphyrin profiles. Patients were classified as type 1 or 2 PCT based on UROD mutation analysis. The CYP1A2*1F promoter A allele frequency was significantly higher (P < 0.022) and the A/A genotype frequency marginally higher in PCT patients overall (P < 0.057), with the A/A genotype significantly more common in type 1 PCT (P < 0.043). The presence of the wild-type GSTM1 allele also was associated significantly with PCT (P < 0.019). Neither hemochromatosis (HFE) mutations, tobacco smoking, hepatitis C and HIV infection, ethanol consumption, nor estrogen use were associated with these allelic variants. Age at onset was significantly lower in type 2 PCT patients (P < 0.001), as observed previously. Thus, positive associations between PCT and the CYP1A2*1F promoter A allele and A/A genotype and the wild-type GSTM1 allele indicates that these functional hepatic biotransformation enzymes are risk factors for the development of this disease.

The effect of bamboo extract on hepatic biotransforming enzymes – Findings from an obese–diabetic mouse model

Bamboo leaves are used as a component in traditional Chinese medicine for the anti-inflammatory function. Our previous studies have demonstrated that an ethanol/water extract from Phyllostachys edulis ameliorated obesity-associated chronic systemic inflammation in mice, and therefore relieving the symptoms of type 2 diabetes. The aim of this project was to further investigate the effects of this bamboo extract on hepatic biotransformation enzymes in both lean and obese mice, as an initial step in the toxicological evaluation of using this traditional medicine in obese/diabetic population. Male C57BL/6J mice were randomized to 4 groups and fed standard (10% kcal from fat) diet with or without bamboo extract supplementation at a dose of 10 gram per kilogram diet (n=10 and n=9, respectively), or high fat (45% kcal from fat) diet with or without bamboo extract (n=8 and N=7, respectively). The dietary treatment lasted for 6 months. Subsequently, the activities and expression of the major Phase I and II hepatic biotransformation enzymes were assessed in subcellular fractions from murine livers. Three groups of mice, lean bamboo extract-supplemented, obese/diabetic, and bamboo extract-supplemented obese/diabetic, showed greater activities of cytochromes P450 1a2 and 3a11 compared to control but no changes in the expression level of these proteins. For Phase II enzymes, bamboo extract supplementation in lean mice caused decreased glutathione-S-transferase activity (-12%) and greater uridine diphosphate glucuronosyltransferase activity (+46%), but had no effect on sulfotransferase activity. Conversely, the obese/diabetic condition itself increased glutathione-S-transferase and uridine diphosphate glucuronosyltransferase activities, but decreased total sulfotransferase activity and sulfotransferase 2a1 expression. Bamboo extract and obesity/diabetes show significant independent effects on hepatic biotransformation as well as interaction effects in mice. These changes may alter the clearance of endo- and xenobiotics, including bamboo extract itself, hence this effect should be carefully considered in the medicinal application of bamboo extract as it has potential to alter its own metabolism and that of other medications concurrently administered to obese diabetic patients.

OSU-A9, a potent indole-3-carbinol derivative, suppresses breast tumor growth by targeting the Akt-NF- B pathway and stress response signaling

The molecular heterogeneity of human tumors challenges the development of effective preventive and therapeutic strategies. To overcome this issue, a rational approach is the concomitant targeting of clinically relevant cellular abnormalities with combination therapy or a potent multi-targeted agent. OSU-A9 is a novel indole-3-carbinol derivative that retains the parent compound's ability to perturb multiple components of oncogenic signaling, but provides marked advantages in chemical stability and antitumor potency. Here, we show that OSU-A9 exhibits two orders of magnitude greater potency than indole-3-carbinol in inducing apoptosis in various breast cancer cell lines with distinct genetic abnormalities, including MCF-7, MDA-MB-231 and SKBR3, with the half maximal inhibitory concentration in the range of 1.2-1.8 microM vis-à-vis 200 microM for indole-3-carbinol. This differential potency was paralleled by OSU-A9's superior activity against multiple components of the Akt-nuclear factor-kappa B (NF-kappaB) and stress response signaling pathways. Notable among these were the increased estrogen receptor (ER)-beta/ERalpha expression ratio, reduced expression of HER2 and CXCR4 and the upregulation of aryl hydrocarbon receptor expression and its downstream target NF-E2 p45-regulated factor (Nrf2). Non-malignant MCF-10A cells were resistant to OSU-A9's antiproliferative effects. Daily oral administration of OSU-A9 at 25 and 50 mg/kg for 49 days significantly inhibited MCF-7 tumor growth by 59 and 70%, respectively, without overt signs of toxicity or evidence of induced hepatic biotransformation enzymes. In summary, OSU-A9 is a potent, orally bioavailable inhibitor of the Akt-NF-kappaB signaling network, targeting multiple aspects of breast tumor pathogenesis and progression. Thus, its translational potential for the treatment or prevention of breast cancer warrants further investigation.

Targeting of the Akt-Nuclear Factor-κB Signaling Network by [1-(4-Chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (OSU-A9), a Novel Indole-3-Carbinol Derivative, in a Mouse Model of Hepatocellular Carcinoma

Constitutive activation of Akt and nuclear factor-kappaB (NF-kappaB) represents major cellular abnormalities associated with the development and progression of hepatocellular carcinoma (HCC). Based on the structure of indole-3-carbinol, a chemopreventive phytochemical, we developed a novel derivative, [1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (OSU-A9), that exhibits higher potency in inducing apoptosis by targeting the Akt-NF-kappaB signaling network. This study was aimed at assessing the antitumor activity of OSU-A9 using both in vitro and in vivo models of HCC, a malignancy in which the Akt-NF-kappaB signaling network plays major roles in pathogenesis and therapeutic resistance. Our data show that OSU-A9 was 100 times more potent than indole-3-carbinol in suppressing the viability of Hep3B, Huh7, and PLC5 HCC cells with IC(50) values ranging from 2.8 to 3.2 microM. OSU-A9 interfered with the interplay between Akt- and NF-kappaB-mediated oncogenic signaling, leading to changes in the functional status of diverse signaling effectors involved in cell cycle progression, apoptosis, angiogenesis, and metastasis. The in vivo efficacy of OSU-A9 was assessed in nude mice bearing luciferase-expressing Hep3B xenograft tumors. Daily oral treatments with OSU-A9 at 25 or 50 mg/kg for 56 days suppressed tumor growth by 67 and 80%, respectively, which was correlated with changes in intratumoral biomarkers pertinent to Akt-NF-kappaB signaling, and without apparent toxicity or evidence of hepatic biotransformation enzyme induction. Together, these findings indicate that OSU-A9 is a potent, orally bioavailable inhibitor of the Akt-NF-kappaB signaling network with a broad spectrum of antitumor activity that includes targets regulating multiple aspects of HCC pathogenesis and progression.