Many chemopreventive agents have encountered bioavailability issues in pre-clinical/clinical studies despite high oral doses. We report here a new concept utilizing polycaprolactone implants embedded with test compounds to obtain controlled systemic delivery, circumventing oral bioavailability issues and reducing the total administered dose. Compounds were released from the implants in vitro dose dependently and for long durations (months), which correlated with in vivo release. Polymeric implants of curcumin significantly inhibited tissue DNA adducts following the treatment of rats with benzo[a]pyrene, with the total administered dose being substantially lower than typical oral doses. A comparison of bioavailability of curcumin given by implants showed significantly higher levels of curcumin in the plasma, liver and brain 30 days after treatment compared with the dietary route. Withaferin A implants resulted in a nearly 60% inhibition of lung cancer A549 cell xenografts, but no inhibition occurred when the same total dose was administered intraperitoneally. More than 15 phytochemicals have been tested successfully by this formulation. Together, our data indicate that this novel implant-delivery system circumvents oral bioavailability issues, provides continuous delivery for long durations and lowers the total administered dose, eliciting both chemopreventive/chemotherapeutic activities. This would also allow the assessment of activity of minor constituents and synthetic metabolites, which otherwise remain uninvestigated in vivo.
Curcumin possess potent anti-inflammatory and anti-proliferative activities but with poor biopharmaceutical attributes. To overcome these limitations, curcumin implants were developed and tissue (plasma, brain and liver) curcumin concentrations were measured in female ACI rats for 3 months. Biological efficacy of tissue levels achieved was analyzed by modulation of hepatic cytochromes. Curcumin implants exhibited diffusion-mediated biphasic release pattern with ~2-fold higher in vivo release as compared to in vitro. Plasma curcumin concentration from implants was ~3.3 ng/ml on day 1 which dropped to ~0.2 ng/ml after 3 months whereas only 0.2–0.3 ng/ml concentration was observed from 4–12 days with diet and was undetected subsequently. Almost 10 fold higher curcumin levels were observed in brain on day 1 from implants compared with diet (30.1±7.3 vs 2.7±0.8 ng/g) and were higher even after 90 days (7.7±3.8 vs 2.2±0.8 ng/g). Although, curcumin levels were similar in liver from both the routes (~25–30 ng/g from day 1–4 and ~10–15 ng/g at 90 days), implants were more efficacious in altering hepatic CYP1A1 levels and CYP3A4 activity at ~28 fold lower doses. Curcumin implants provided much higher plasma and tissue concentrations and are a viable alternative for delivery of curcumin to various organs like brain.
Curcumin; Implants; Bioavailability; Chemoprevention; Controlled Release; Tissue curcumin levels
Adenosine (AD) elicits cardioprotection through A1-receptor (A1R) activation. Therapy with AD A1R agonists, however, is limited by undesirable actions of full agonism such as bradycardia. This study examined the effects of capadenoson (CAP), a partial AD A1R agonist, on left ventricular (LV) function and remodeling in dogs with heart failure (HF).
Methods and Results
12 dogs with microembolization-induced HF were randomized to 12 weeks oral therapy with CAP (7.5 mg Bid, n=6) or to no therapy (Control, n=6). LV end-diastolic (EDV) and end-systolic (ESV) volumes, ejection fraction (EF), plasma norepinephrine (NE) and n-terminal pro-brain natriuretic peptide (nt-pro BNP) were measured before (PRE) and 1 and 12 weeks after therapy (POST). LV tissue obtained at POST was used to assess volume fraction of interstitial fibrosis (VFIF), SERCA-2a activity, expression of mitochondria uncoupling proteins (UCP) and glucose transporters (GLUT). In controls, EDV and ESV increased and EF decreased significantly from PRE to POST (EF: 30±2 vs. 27±1 %, p<0.05). In CAP-treated dogs, EDV was unchanged; EF increased significantly after one week (36±2 vs. 27±2 %, p<0.05) with a further increase at POST (39±2 %, p<0.05) while ESV decreased. CAP significantly decreased VFIF, normalized SERCA-2a activity and expression of UCP-2 and -3, and GLUT-1 and -2 and significantly decreased NE and nt-pro BNP.
In HF dogs, CAP improves LV function and prevents progressive remodeling. Improvement of LV systolic function occurs early after initiating therapy. The results support development of partial AD A1R agonists for the treatment of chronic HF.
Heart failure; Ventricular remodeling; Protein expression; Adenosine receptors
Chronic heart failure (HF) is associated with autonomic dysregulation characterized by a sustained increase of sympathetic drive and by withdrawal of parasympathetic activity. Sympathetic overdrive and increased heart rate are predictors of poor long-term outcome in patients with HF. Considerable evidence exists that supports the use of pharmacologic agents that partially inhibit sympathetic activity as effective long-term therapy for patients with HF; the classic example is the wide use of selective and non-selective beta-adrenergic receptor blockers. In contrast, modulation of parasympathetic activation as potential therapy for HF has received only limited attention over the years given its complex cardiovascular effects. In this article, we review results of recent experimental animal studies that provide support for the possible use of electrical Vagus nerve stimulation (VNS) as a long-term therapy for the treatment of chronic HF. In addition to exploring the effects of chronic VNS on left ventricular (LV) function, the review will also address the effects of VNS on potential modifiers of the HF state that include cytokine production and nitric oxide elaboration. Finally, we will briefly review other nerve stimulation approaches also currently under investigation as potential therapeutic modalities for treating chronic HF.
Ventricular function; Ventricular remodeling; Electrical nerve stimulation; Sympathetic activity; Parasympathetic activity; Animal models of disease; Cytokines; Nitric oxide
Exposure to cigarette smoke is well documented to increase oxidative stress and could account for higher risk of cervical cancer in smokers. Cervical pre-cancerous lesions that are initiated by human papillomavirus (HPV) infection generally regress in the absence of known risk factors such as smoking. 8-oxodeoxyguanosine (8-oxodG) is a highly mutagenic oxidative DNA lesion that is formed by the oxidation of deoxyguanosine. In the present study, we examined: a) the effect of cigarette smoke condensate (CSC) on 8-oxodG formation in and its removal from HPV-transfected (ECT1/E6 E7), HPV-positive (CaSki) and HPV-negative (C33A) human cervical cancer cells, and b) the cell cycle progression and apoptosis in CSC-treated ECT1/E6 E7 cells. CSC induced 8-oxodG in a dose-(p=0.03) and time (p=0.002)-dependent fashion in ECT1/E6 E7 cells as determined by flow cytometry. A 2.4-fold higher level of 8-oxodG was observed in HPV-positive compared with HPV-negative cells. However, 8-oxodG lesions were almost completely removed 72 h post-exposure in all cell lines as determined by ImageStream analysis. This observation correlates with the 2- and 5-fold increase in the p53 levels in ECT1/E6 E7 and CaSki cells with no significant change in C33A cells. We conclude that: a) cigarette smoke constituents induce oxidative stress with higher burden in HPV-positive cervical cancer cells and b) the significant increase observed in p53 levels in wild-type cervical cells (ECT1/E6 E7 and CaSki) may be attributed to the p53-dependent DNA repair pathway while a p53-independent pathway in C33A cells cannot be ruled out.
cigarette smoke condensate; human cervical cancer cells; HPV; oxidative DNA damage; DNA repair
In recent years, there has been a significant improvement in the understanding of molecular events and critical pathways involved in breast cancer. This has led to the identification of novel targets and development of anticancer therapies referred to as targeted therapy. Targeted therapy has high specificity for the molecules involved in key molecular events that are responsible for cancer phenotype such as cell growth, survival, migration, invasion, metastasis, apoptosis, cell-cycle progression, and angiogenesis. Targeted agents that have been approved for breast cancer include trastuzumab and lapatinib, directed against human epidermal growth factor receptor 2 (HER2) and bevacizumab, directed against vascular endothelial growth factor (VEGF). Several other targeted agents currently under evaluation in preclinical and clinical trials include inhibitors of epidermal growth factor receptor (EGFR), dual EGFR and HER2 inhibitors, VEGF/VEGFR inhibitors, and agents that interfere with crucial signaling pathways such as PI3K/AKT/mTOR and RAS/MEK/ERK; agents against other tyrosine kinases such as Src, insulin-like growth factor (IGF)/IGF-receptor (IGFR); agents that promote apoptosis such as Poly ADP ribose polymerase inhibitors; agents that target invasion and metastasis such as matrix metalloproteinases inhibitors and others. In this review, we highlight the most promising targeted agents and their combination with mainstream chemotherapeutic drugs in clinical trials.
Breast cancer; chemotherapy; targeted therapy
Colored fruits, particularly berries, are highly chemoprotective because of their antioxidant, anti-proliferative and anti-inflammatory activities. We report cancer chemoprotective potential of Syzygium cumini L., commonly known as ‘jamun’ or Indian blackberry. Anthocyanins and other polyphenolics were extracted with acidic ethanol, and enriched by amberlite XAD7/HP20 (1:1). The pulp powder was found to contain 0.54% anthocyanins, 0.17% ellagic acid/ellagitannins and 1.15% polyphenolics. Jamun seed contained no detectable anthocyanins, but had higher amounts of ellagic acid/ellagitannins (0.5%) and total polyphenolics (2.7%) than the pulp powder. Upon acid hydrolysis, the pulp extract yielded five anthocyanidins by HPLC: malvidin (44.4%), petunidin (24.2%), delphinidin (20.3%), cyanidin (6.6%), and peonidin (2.2%). Extracts of both jamun pulp (1,445±64 μmol of trolox equivalent (TE)/g) and seeds (3,379±151 μM of TE/g) showed high oxygen radical absorbance capacity (ORAC). Their high antioxidant potential was also reflected by 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)- and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging, and ferrous ion-chelating activities. We also analyzed anti-proliferative activity of jamun extracts against human lung cancer A549 cells. The hydrolyzed pulp and seed extracts showed significant antiproliferative activity. However, unhydrolyzed extracts showed much less activity. These data showed that in addition to five anthocyanidins, jamun contains appreciable amounts of ellagic acid/ellagitannins, with high antioxidant and antiproliferative activities.
Anthocyanins; Syzygium cumini; ellagic acid and ellagitannins; antioxidant activity; anti-proliferative activity
We performed a structure-activity relationship (SAR) study of a novel aspirin (ASA) derivative, which shows strong anticancer activity in vitro and in vivo. A series of ASA-based benzyl esters (ABEs) were synthesized and their inhibitory activity against human colon (HT-29 and SW480) and pancreatic (BxPC-3 and MIA PaCa-2) cancer cell lines was evaluated. The ABEs that we studied largely comprise organic benzyl esters bearing an ASA or acyloxy group (X) at the meta or para position of the benzyl ring and one of four different leaving groups. The nature of the salicyloyl/acyloxy function, the leaving group, and the additional substituents affecting the electron density of the benzyl ring, all were influential determinants of the inhibitory activity on cancer cell growth for each ABE. Positional isomerism also played a significant role in this effect. The mechanism of action of these compounds appears consistent with the notion that they generate either a quinone methide or an m-oxybenzyl zwitterion (or an m-hydroxybenzyl cation), which then reacts with a nucleophile, mediating their biological effect. Our SAR study provides an insight into the biological properties of this novel class of compounds and underscores their potential as anticancer agents.
anticancer compounds; phosphoaspirin; nonsteroidal anti-inflammatory drugs; cancer prevention
Exposure to excessive manganese (Mn) levels leads to neurotoxicity, referred to as manganism, which resembles Parkinson’s disease (PD). Manganism is caused by neuronal injury in both cortical and subcortical regions, particularly in the basal ganglia. The basis for the selective neurotoxicity of Mn is not yet fully understood. However, several studies suggest that oxidative damage and inflammatory processes play prominent roles in the degeneration of dopamine-containing neurons. In the present study, we assessed the effects of Mn on reactive oxygen species (ROS) formation, changes in high-energy phosphates and associated neuronal dysfunctions both in vitro and in vivo. Results from our in vitro study showed a significant (P<0.01) increase in biomarkers of oxidative damage, F2-isoprostanes (F2-IsoPs), as well as the depletion of ATP in primary rat cortical neurons following exposure to Mn (500 µM) for 2 hours. These effects were protected when neurons were pretreated for 30 min with 100 µM of an antioxidant, the hydrophilic vitamin E analog, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), or an anti-inflammatory agent, indomethacin. Results from our in vivo study confirmed a significant increase in F2-IsoPs levels in conjunction with the progressive spine degeneration and dendritic damage of the striatal medium spiny neurons (MSNs) of mice exposed to Mn (100 mg/kg, s.c.) 24 hours. Additionally, pretreatment with vitamin E (100 mg/kg, i.p.) or ibuprofen (140 µg/ml in the drinking water for two weeks) attenuated the Mn-induced increase in cerebral F2-IsoPs and protected the MSNs from dendritic atrophy and dendritic spine loss. Our findings suggest that the mediation of oxidative stress/mitochondrial dysfunction and the control of alterations in biomarkers of oxidative injury, neuroinflammation and synaptodendritic degeneration may provide an effective, multi-pronged therapeutic strategy for protecting dysfunctional dopaminergic transmission and slowing of the progression of Mn-induced neurodegenerative processes.
Manganese; oxidative stress; medium spiny neurons; neurodegeneration; vitamin E; trolox; ibuprofen
From ancient times, chemopreventive agents have been used to treat/prevent several diseases, including cancer. They are found to elicit a spectrum of potent responses including anti-inflammatory, anti-oxidant, anti-proliferative, anti-carcinogenic, and anti-angiogenic activity in various cell culture and some animal studies. Research over the past four decades has shown that chemopreventives affect a number of proteins involved in various molecular pathways that regulate inflammatory and carcinogenic responses in a cell. Various enzymes, transcription factors, receptors, and adhesion proteins are also affected by chemopreventives. Although, these natural compounds have shown significant efficacy in cell-culture studies, they elicited limited efficacy in various clinical studies. Their introduction into the clinical setting is hindered largely by their poor solubility, rapid metabolism, or a combination of both, ultimately resulting in poor bioavailability upon oral administration. Therefore, to circumvent these limitations and to ease their transition to clinics, alternate strategies should be explored. Drug delivery systems such as nanoparticles, liposomes, microemulsions, and polymeric implantable devices are emerging as one of the viable alternatives that have been demonstrated to deliver therapeutic concentrations of various potent chemopreventives such as curcumin, ellagic acid, green tea polyphenols, and resveratrol into the systemic circulation. In this review article, we have attempted to provide a comprehensive outlook for these delivery approaches, using curcumin as a model agent, and discussed future strategies to enable the introduction of these highly potent chemopreventives into a physician’s armamentarium.
Chemoprevention; Curcumin; Drug Delivery; Bioavailability; Antioxidant
The polyphenolics in green tea are believed to be the bioactive components. However, poor bioavailability following ingestion limits their efficacy in vivo. In this study, polyphenon E (poly E), a standardized green tea extract, was administered by sustained-release polycaprolactone implants (two, 2-cm implants; 20% drug load) grafted subcutaneously or via drinking water (0.8% w/v) to female S/D rats. Animals were treated with continuous low dose of benzo[a]pyrene (BP) via subcutaneous polymeric implants (2 cm; 10% load) and euthanized after 1 and 4 weeks. Analysis of lung DNA by 32P-postlabeling resulted in a statistically significant reduction (50%; p=0.023) of BP-induced DNA adducts in the implant group; however, only a modest (34%) but statistically insignificant reduction occurred in the drinking water group at 1 week. The implant delivery system also showed significant reduction (35%; p=0.044) of the known BP diolepoxide-derived DNA adduct after 4 weeks. Notably, the total dose of poly E administered was >100-fold lower in the implant group than the drinking water group (15.7 versus 1,632 mg, respectively). Analysis of selected phase I, phase II, and nucleotide excision repair enzymes at both mRNA and protein levels showed no significant modulation by poly E, suggesting that the reduction in the BP-induced DNA adducts occurred presumably due to known scavenging of the anti-diolepoxide of BP by the poly E catechins. In conclusion, our study demonstrated that sustained systemic delivery of poly E significantly reduced BP-induced DNA adducts in spite of its poor bioavailability following oral administration.
Green tea polyphenols; Polymeric implants; Plasma levels of EGCG; Benzo[a]pyrene; Sustained P450 levels; 32P-Postlabeling
Dietary habits that expose populations to potential toxicants as well as protective agents simultaneously is a realistic scenario where a meaningful assessment of the interactions and net benefit or damage can be made. A group of Inuit from Salluit, Northern Canada are exposed to high levels of PCBs and selenium, both present in the Inuit traditional foods such as blubber from sea mammals and fatty fish. Blood samples were collected from 83 Inuit, 22–70 years old. Blood selenium and PCB levels were determined previously and ranged from 227 to 2,069 µg/L and 1.7 to 143 µg/L, respectively. DNA isolated from white blood cells were analyzed by modified 32P-postlabeling adductomics technology that detects a multitude of highly polar to lipophilic adducts. The levels of 8-oxodG adducts ranged from 470 to 7,400 adducts/109 nucleotides. Other as yet unidentified polar adducts showed a 30 to 800–fold inter-individual variability. Adduct levels were negatively associated with PCB and selenium levels. The subjects were classified into high and low ratio groups, with respect to selenium/PCB. In the high ratio group, the coefficient of selenium is significantly negatively correlated with 8-oxodG (r = −0.38, p = 0.014) and total adducts (r = −0.41, p = 0.009) while there was no correlation within the low selenium/PCB group. This study suggests increasing selenium has mitigating effect in reducing DNA adducts and therefore, possible negative effects of PCB were not rendered. A protective effect of selenium is highlighted.
Selenium; PCBs; Inuit; 8-oxodG; DNA adducts; 32P-postlabeling
There is increasing evidence supporting a causal role of oxidatively damaged DNA in neurodegeneration during the natural aging process and neurodegenerative diseases such as Parkinson’s and Alzheimer’s. The presence of redox-active catecholamine neurotransmitters coupled with the localization of catalytic copper to DNA suggests a plausible role for these agents in the induction of oxidatively generated DNA damage. In this study we have investigated the role of Cu(II)-catalyzed oxidation of several catecholamine neurotransmitters and related neurotoxins to induce oxidatively generated DNA damage. Auto-oxidation of all catechol neurotransmitters and related congeners tested resulted in the formation of nearly a dozen oxidation DNA products resulting in a decomposition pattern that was essentially identical for all agents tested. The presence of Cu(II), and to a lesser extent Fe(III), had no effect on the decomposition pattern but substantially enhanced the DNA product levels by up to 75 fold, with dopamine producing the highest levels of unidentified oxidation DNA products (383 ± 46 adducts/106 nucleotides), comparable to 8-oxo-7,8-dihydro-2′-deoxyguanosine levels under the same conditions (122 ± 19 adducts/106 nucleotides). The addition of sodium azide, 2,2,6,6-tetramethyl-4-piperidone, tiron, catalase, bathocuproine or methional to the dopamine/Cu(II) reaction mixture resulted in a substantial decrease (>90%) in oxidation DNA product levels, indicating a role of singlet oxygen, superoxide, H2O2, Cu(I) and Cu(I)OOH in their formation. While the addition of N-tert-butyl-α-phenylnitrone significantly decreased (67%) dopamine-mediated oxidatively damaged DNA, three other hydroxyl radical scavengers, ascorbic acid, sodium benzoate and mannitol, had little to no effect on these oxidation DNA product levels, suggesting that free hydroxyl radicals may have limited involvement in this dopamine/Cu(II)-mediated oxidatively generated DNA damage. These studies suggest a possible contributory role of oxidatively generated DNA damage by dopamine and related catechol neurotransmitters/neurotoxins in neurodegeneration and cell death. We also found that a naturally occurring broad spectrum antioxidant, ellagic acid, was substantially effective (nearly 50% inhibition) at low doses (1 μM) at preventing this dopamine/Cu(II)-mediated oxidatively generated DNA damage. Since dietary ellagic acid has been found to reduce oxidative stress in rat brains, a neuro-protective role of this polyphenol is plausible.
Neurotransmitters; Dopamine; Catecholamines; Oxidative DNA adducts; 32P-Postlabeling
Autonomic abnormalities exist in heart failure (HF) and contribute to disease progression. Activation of the Carotid sinus baroreflex (CSB) has been shown to reduce sympathetic outflow and augment parasympathetic vagal tone. This study tested the hypothesis that long-term electrical activation of carotid sinus baroreflex improves left ventricular (LV) function and attenuates progressive LV remodeling in dogs with advanced chronic HF.
Methods and Results
Studies were performed in 14 dogs with coronary microembolization-induced HF (LV ejection fraction, EF ~25%). Eight dogs were chronically instrumented for bilateral CSB activation using the Rheos® System (CVRx® Inc., Minneapolis, MN) and 6 were not and served as controls. All dogs were followed for 3 months and none received other background therapy. During follow-up, treatment with CSB increased LV EF 4.0 ± 2.4 % compared to a reduction in control dogs of −2.8 ± 1.0% (p<0.05). Similarly, treatment with CSB decreased LV end-systolic volume −2.5 ± 2.7 ml compared to an increase in control dogs of 6.7 ± 2.9 ml (p<0.05). Compared to control, CSB activation significantly decreased LV end-diastolic pressure and circulating plasma norepinephrine, normalized expression of cardiac β1-adrenergic receptors, β-adrenergic receptor kinase and nitric oxide synthase and reduced interstitial fibrosis and cardiomyocyte hypertrophy.
In dogs with advanced HF, CSB activation improves global LV function and partially reverses LV remodeling both globally and at cellular and molecular levels.
heart failure; ventricular remodeling; gene expression; baroreflex function
In hypertensive subjects, angiotensin II and endothelin participate in a manner involving closely interwoven pathways in increasing blood pressure (BP) and inducing end organ damage. The primary objective of this study was to determine the effect of TRC120038, a novel dual AT1/ETA receptor blocker on BP, in obese Zucker spontaneously hypertensive fatty rats (ob-ZSF1), an animal model of moderate hypertension, diabetes with progressive renal and cardiac dysfunction. Ob-ZSF1 rats loaded with 0.5% salt were treated with TRC120038 (11.8 mg/kg bid.) or candesartan cilexetil (0.3 mg/kg od.) or vehicle control. Blood pressure (by radio-telemetry) and renal functional markers were monitored throughout the study. Cardiac function was assessed terminally by pressure volume catheter. Markers for renal dysfunction were measured and changes were evaluated histopathologically. TRC120038 showed greater fall in both systolic and diastolic BP in comparison to candesartan at its maximum antihypertensive dose. TRC120038 also reduced the severity of renal dysfunction and preserved cardiac function in ob-ZSF1 rat.
Background & Aims
Non-steroidal anti-inflammatory drugs (NSAIDs) are effective cancer chemopreventive agents. However, chronic administration of NSAIDs is associated with significant side effects, mainly gastrointestinal. Given these limitations, we synthesized phospho-sulindac (P-S; OXT-328), a novel sulindac derivative.
Here, we evaluated the safety and efficacy of P-S in preclinical models, including its mechanism of action using human colon cancer cell (HCCC) lines and animal tumor models.
a) Compared to sulindac, P-S is much more potent in inhibiting the growth of cultured HCCC and more efficacious in preventing the growth of HT-29 xenografts in nude mice. P-S also prevents the growth of intestinal tumors in Apc/Min mice, b) in combination with difluoromethylornithine (DFMO), P-S reduced tumor multiplicity in Apc/Min mice by 90%; and c) P-S is much safer than sulindac as evidenced by its in vitro toxicological evaluation and animal toxicity studies. Mechanistically, P-S increases the intracellular levels of reactive oxygen and nitrogen species, which are key early mediators of its chemopreventive effect. Moreover, P-S induces spermidine/spermine N1-acetyltransferase enzymatic activity, and together with DFMO it reduces polyamine levels in vitro and in vivo.
P-S displays considerable safety and efficacy, two pharmacological properties that are essential for a potential cancer chemopreventive agent, and thus merits further evaluation.
Colon cancer; Phospho-sulindac; Sulindac; Polyamines; Reactive oxygen species
In order to determine whether dietary berries and ellagic acid prevent 17β estradiol (E2) -induced mammary tumors by altering estrogen metabolism, we randomized ACI rats (n=6/group) into 5 groups − sham implant + control diet (SH-CD), E2 − implant + control diet (E2-CD), E2+2.5% black raspberry (E2-BRB); E2+2.5% blueberry (E2-BB) and E2+ 400ppm ellagic acid (E2-EA). Animals were euthanized at early (6wk), intermediate (18wk) and late (24wk) phases of E2-carcinogenesis and the mammary tissue analyzed for gene-expression changes using quantitative real-time PCR. At 6 weeks, E2-treatment caused 48-fold increase in cytochrome P4501A1(CYP1A1) (p<0.0001), which was attenuated by both BRB and BB diets to 12- and 21-fold, respectively (p<0.001). E2 did not alter CYP1B1 levels, but both berry and EA diets significantly suppressed it by 11- and 3.5-fold, respectively from baseline (p<0.05). There was a 5-fold increase in 17β-Hydroxysteroid dehyrdogenase(17βHSD7) and this was moderately abrogated to about 2-fold by all supplementation (p<0.05). At 18 weeks, CYP1A1 was elevated by 15-fold in E2-CD and only E2-BB reduced this increase to 7-fold (p<0.05). Catechol-O-methyl transferase(COMT) expression was elevated 2-fold by E2-treatment (p<0.05) and all supplementation reversed this. At 24 weeks, CYP1A1 expression was less pronounced, but still high (8-fold) in E2-treated rats. This increase was reduced to 3.2 and 4.6-fold, by E2-BRB and E2-EA, respectively (p<0.05), but not by E2-BB. Supplementation did not alter the effect of E2 on steroid receptors. The diets also significantly suppressed mammary tumor incidence (10–30%), volume (41–67%) and multiplicity (38 to 51%) (p<0.05). Berries may prevent mammary tumors by suppressing the levels of E2-metabolizing enzymes during the early phase of E2-carcinogenesis.
Estrogen; ACI rats; berries; polyphenols; breast cancer prevention; CYP450
Lung cancer is the leading cause of cancer deaths in the U.S., surpassing breast cancer as the primary cause of cancer-related mortality in women. The goal of the present study was to identify early molecular changes in the lung induced by exposure to tobacco smoke and thus identify potential targets for chemoprevention. Female A/J mice were exposed to either tobacco smoke or HEPA-filtered air via a whole-body exposure chamber (6 h/day; 5 days/wk for 3, 8 and 20 wk). Gene expression profiles of lung tissue from control and smoke-exposed animals were established using a 15 K cDNA microarray. Cytochrome P450 1b1 (Cyp1b1), a Phase I enzyme involved in both the metabolism of xenobiotics and the 4-hydroxylation of 17β-estradiol, was modulated to the greatest extent following smoke exposure. A panel of 10 genes was found to be differentially expressed in control and smoke-exposed lung tissue at 3, 8 and 20 wk (P < 0.001). The interaction network of these differentially expressed genes revealed new pathways modulated by short-term smoke exposure including estrogen metabolism. In addition, 17β-estradiol was detected within murine lung tissue by gas chromatography coupled mass spectrometry and immunohistochemistry. Identification of the early molecular events that contribute to lung tumor formation is anticipated to lead to the development of promising targeted chemopreventive therapies. In conclusion, the presence of 17β-estradiol within lung tissue when combined with the modulation of Cyp1b1 and other estrogen metabolism genes by tobacco smoke provides novel insight into a possible role for estrogens in lung cancer.
Tobacco smoke; mouse lung; gene expression; Cyp1b1; estrogen
Chronic overnutrition and consequential visceral obesity is associated with a cluster of risk factors for cardiovascular disease and type 2 diabetes mellitus. Moreover, individuals who have a triad of hypertension, dysglycemia, and elevated triglycerides along with reduced high-density lipoprotein cholesterol have a greater residual cardiovascular risk even after factoring for the traditional risk factors such as age, smoking, diabetes, and elevated low-density lipoprotein cholesterol. In our previous study we demonstrated that TRC150094, when administered to rats receiving a high-fat diet, stimulated mitochondrial fatty acid oxidation (FAO) and reduced visceral adiposity, opening an interesting perspective for a possible clinical application. In the present study, oral administration of TRC150094 to obese Zucker spontaneously hypertensive fatty rats (obese ZSF1) improved glucose tolerance and glycemic profile as well as attenuated a rise in blood pressure. Obese ZSF1 rats treated with TRC150094 also showed reduced hepatic steatosis, reduced progression of nephropathy, and improved skeletal muscle function. At the cellular level, TRC150094 induced a significant increase in mitochondrial respiration as well as an increased FAO in liver and skeletal muscle, ultimately resulting in reduced hepatic as well as total body fat accumulation, as evaluated by magnetic resonance spectroscopy and magnetic resonance imaging, respectively. If reproduced in humans, these results could confirm that TRC150094 may represent an attractive therapeutic agent to counteract multiple residual cardiovascular risk components.
CV risk factors; energy expenditure; fatty acid oxidation; obesity; TRC150094; type 2 diabetes
Human papillomavirus (HPV) is the causative factor in the development and progression of cervical cancers in >97% of the cases, although insufficient. Epidemiological studies suggest an elevated risk of cervical cancer for cigarette smokers; therefore, we examined cigarette smoke-induced DNA damage and repair in HPV16-transformed human ectocervical cells (ECT1/E6 E7). Cells were treated with cigarette smoke condensate (CSC) for 72 h to assess the formation of single- and double-strand DNA breaks, measured by alkaline and neutral single cell gel electrophoresis assays, respectively. The mean tail length of cells with single-strand breaks was increased by 1.8-, 2.7- and 3.7-fold (p<0.001) after treatment with 4, 8 and 12 µg/ml CSC, respectively. The tail length with double-strand breaks was also increased dose-dependently. These results were further supported by measurement of the mean tail moment: the increase in both single- and double-strand breaks were much more pronounced with increasing concentration of CSC, by up to 23.5-fold (p<0.0001 for both assays). To examine the DNA repair, cells were treated with CSC for 72 h, followed by CSC withdrawal and re-incubation of the cells with fresh medium for 24, 48, or 72 h. Both single- and double-strand DNA breaks were removed during the initial 24 h but no further removal of the damage was observed. Up to 80% of residual single- and double-strand DNA breaks (p<0.05) were found to persist at all CSC concentrations examined. Ellagic acid, a known antioxidant and free-radical scavenger, was found to significantly inhibit DNA breaks induced by CSC. Thus, free radicals may be a plausible source of CSC-induced DNA damage. These data show that CSC-mediated DNA strand breaks are highly persistent, and suggest that persistence of cigarette smoke-associated DNA damage in the presence of HPV infection may lead to increased mutations in cervical cells and ultimately higher cancer risk.
cervical cancer; human papillomavirus; cigarette smoke condensate; comet assay; DNA strand breaks; ellagic acid
Prophylactic agents acutely administered in response to anticholinesterases intoxication can prevent toxic symptoms, including fasciculations, seizures, convulsions and death. However, anticholinesterases also have long-term unknown pathophysiological effects, making rational prophylaxis/treatment problematic. Increasing evidence suggests that in addition to excessive cholinergic stimulation, organophosphate compounds such as diisopropylphosphorofluoridate (DFP) induce activation of glutamatergic neurons, generation of reactive oxygen (ROS) and nitrogen species (RNS), leading to neurodegeneration. The present study investigated multiple affectors of DFP exposure critical to cerebral oxidative damage and whether antioxidants and NMDA receptor antagonist memantine provide neuroprotection by preventing DFP-induced biochemical and morphometric changes in rat brain. Rats treated acutely with DFP (1.25 mg/kg, s.c.) developed onset of toxicity signs within 7-15 min that progressed to maximal severity of seizures and fasciculations within 60 min. At this time point, DFP caused significant (p<0.01) increases in biomarkers of ROS (F2-isoprostanes, F2-IsoPs; and F4-neuroprostanes, F4-NeuroPs), RNS (citrulline), and declines in high-energy phosphates (HEP) in rat cerebrum. At the same time, quantitative morphometric analysis of pyramidal neurons of the hippocampal CA1 region revealed significant (p<0.01) reductions in dendritic lengths and spine density. When rats were pretreated with the antioxidants N-tert-butyl-α-phenylnitrone (PBN, 200 mg/kg, i.p.), or vitamin E (100 mg/kg, i.p./day for 3 days), or memantine (18 mg/kg, i.p.), significant attenuations in DFP-induced increases in F2-IsoPs, F4-NeuroPs, citrulline, and depletion of HEP were noted. Furthermore, attenuation in oxidative damage following antioxidants or memantine pretreatment was accompanied by rescue from dendritic degeneration of pyramidal neurons in the CA1 hippocampal area. These findings closely associated DFP-induced lipid peroxidation with dendritic degeneration of pyramidal neurons in the CA1 hippocampal area and point to possible interventions to limit oxidative injury and dendritic degeneration induced by anticholinesterase neurotoxicity.
Exposure to excessive manganese (Mn) levels results in neurotoxicity to the extrapyramidal system and the development of Parkinson’s disease (PD)-like movement disorder, referred to as manganism. Although the mechanisms by which Mn induces neuronal damage are not well defined, its neurotoxicity appears to be regulated by a number of factors, including oxidative injury, mitochondrial dysfunction and neuroinflammation. To investigate the mechanisms underlying Mn neurotoxicity, we studied the effects of Mn on reactive oxygen species (ROS) formation, changes in high-energy phosphates (HEP), neuroinflammation mediators and associated neuronal dysfunctions both in vitro and in vivo. Primary cortical neuronal cultures showed concentration-dependent alterations in biomarkers of oxidative damage, F2-isoprostanes (F2-IsoPs) and mitochondrial dysfunction (ATP), as early as 2 hours following Mn exposure. Treatment of neurons with 500 µM Mn also resulted in time-dependent increases in the levels of the inflammatory biomarker, prostaglandin E2 (PGE2). In vivo analyses corroborated these findings, establishing that either a single or three (100 mg/kg, s.c.) Mn injections (days 1, 4 and 7) induced significant increases in F2-IsoPs and PGE2 in adult mouse brain 24 hours following the last injection. Quantitative morphometric analyses of Golgi-impregnated striatal sections from mice exposed to single or three Mn injections revealed progressive spine degeneration and dendritic damage of medium spiny neurons (MSNs). These findings suggest that oxidative stress, mitochondrial dysfunction and neuroinflammation are underlying mechanisms in Mn-induced neurodegeneration.
Manganese; oxidative stress; medium spiny neurons; neurodegeneration; spines; manganism
Polychlorinated biphenyls (PCBs) are toxic industrial chemicals, complete carcinogens and efficacious tumor promoters. However, the mechanism(s) of PCB-mediated carcinogenicity remains largely undefined. One likely pathway by which these agents may play a role in carcinogenesis is the generation of oxidative DNA damage by redox cycling of dihydroxylated PCB metabolites. We have now employed a new 32P-postlabeling system to examine novel oxidative DNA lesions induced by Cu2+-mediated activation of PCB metabolites. 32P-Postlabeling of DNA incubated with various PCB metabolites resulted in over a dozen novel polar oxidative DNA adducts that were chromatographically similar for all active agents. The most potent metabolites tested were the hydroquinones (hydroxyl groups arranged para to each other) yielding polar oxidative adduct levels ranging from 55 to 142 adducts/106 nucleotides. PCB catechols, or ortho-dihydroxy metabolites, were up to 40% less active than their corresponding hydroquinone congeners while mono hydroxylated and quinone metabolites did not produce detectable oxidative damage over that of vehicle. With the exception of 2,4,5-Cl-2′,5′-dihydroxybiphenyl, this oxidative DNA damage appeared to be inversely related to chlorine content: no chlorine ≈ mono- > di- > tri-chlorinated metabolites. Importantly, copper, but not iron, was essential for activation of the PCB metabolites to these polar oxidative DNA adducts since in its absence or in the presence of the Cu+-specific scavenger, bathocuproine, no adducts were detected. Intervention studies with known reactive oxygen species (ROS) modifiers suggested that H2O2, singlet oxygen, hydroxyl radical and superoxide may also be involved in this PCB-mediated oxidative DNA damage. These data indicate a mechanistic role of several ROS, in addition to copper, in PCB-induced DNA damage and provide further support for oxidative DNA damage in PCB-mediated carcinogenesis.
PCBs; oxidative DNA adducts; 32P-postlabeling
β-Blockers are standard therapy for patients with heart failure (HF). This study compared the effects of chronic monotherapy with 2 different β1-selective adrenoceptor blockers, namely atenolol and metoprolol succinate, on left ventricular (LV) function and remodeling in dogs with coronary microembolization-induced HF [LV ejection fraction (EF) 30–40%].
Twenty HF dogs were randomized to 3 months of therapy with atenolol (50 mg once daily, n = 6), metoprolol succinate (100 mg, once daily, n = 7) or to no therapy (control, n = 7). LV EF and volumes were measured before initiating therapy and after 3 months of therapy. The change (Δ) in EF and volumes between measurements before and after therapy was calculated and compared among study groups.
In controls, EF decreased and end-systolic volume increased. Atenolol prevented the decrease in EF and the increase in ESV. In contrast, metoprolol succinate significantly increased EF and decreased end-systolic volume. ΔEF was significantly higher and Δend-systolic volume significantly lower in metoprolol succinate-treated dogs compared to atenolol-treated dogs (EF: 6.0 ± 0.86% vs. 0.8 ± 0.85%, p < 0.05; end-systolic volume: −4.3 ± 0.81 ml vs. −1 ± 0.52 ml, p <0.05).
In HF dogs, chronic therapy with atenolol does not elicit the same LV function and remodeling benefits as those achieved with metoprolol succinate.
Heart failure; Myocyte hypertrophy; Ventricular remodeling; Gene expression
Colon cancer is second leading cause of cancer-related deaths in Western countries. Diet and smoking, which contain aromatic and heterocyclic amines, are major risk factors for colon cancer. Colorectal cancers have a natural history of long latency and therefore provide ample opportunities for effective chemoprevention. 3,2’-Dimethyl-4-aminobiphenyl (DMABP) is an experimental aromatic amine that causes cancer in rat colon and serves as an experimental model for arylamine and heterocyclic amine mutagens derived from diet and smoking. In this study, we investigated the effects of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor on DMABP-induced DNA adduct formation in rat liver and colon. Male F-344 rats (5-weeks old) were provided free access to modified AIN-76A rat chow containing 0 (control), 500, 1000, or 1500 ppm celecoxib. Two weeks later, the rats received a subcutaneous injection of 100 mg/kg DMABP in peanut oil. Two days after DMABP treatment, the rats were killed and DMABP-derived adducts were analyzed in colon and liver DNA by butanol extraction-mediated 32P-postlabeling. Two major DNA adducts, identified as dG-C8-DMABP and dG-N2-DMABP, were detected in liver and colon of rats treated with DMABP. These DNA adducts were diminished approximately 35–40% with 500 ppm and 65–70% with 1,000 ppm celecoxib. In the colon, no further decline in DNA adducts was observed at 1500 ppm. The same DMABP-DNA adducts also were detected in the liver and were also diminished by celecoxib treatment. The reduction in DMABP-DNA adduct levels in celecoxib-treated animals provides further support for celecoxib as a chemopreventive agent for colorectal cancer.
3,2’-Dimethyl-4-aminobiphenyl; DNA adducts; celecoxib; colon cancer; 32P-postlabeling