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1.
J Invest Dermatol ; 2024 Jun 13.
Article in English | MEDLINE | ID: mdl-38897541

ABSTRACT

RAS proteins regulate cell division, differentiation and apoptosis via multiple downstream effector pathways. Oncogenic RAS variants are the commonest drivers in cancers, however they also drive many benign lesions predisposing to malignancy, such as melanocytic naevi, thyroid nodules, and colonic polyps. Reversal of these benign lesions could reduce cancer incidence, however the effects of oncogenic RAS have been notoriously difficult to target with downstream pathway inhibitors. Here we show effective suppression of oncogenic and currently undruggable NRASQ61K in primary cells from melanocytic naevi using siRNA targeted to the recurrent causal variant. This results in striking reduction in expression of ARL6IP1, a known inhibitor of endoplasmic reticulum stress-induced apoptosis not previously linked to NRAS. We go on to show that a single dose of siRNA in primary cells triggers an apoptotic cascade, in contrast to treatment with a MEK inhibitor. Protective packaging of the targeted siRNA into lipid nanoparticles permits successful delivery into a humanised mouse model of melanocytic naevi, and results in variant NRAS knockdown in vivo. These data show that RAS-induced protection from apoptosis is involved in persistence of NRAS-driven melanocytic naevi and anticipate that targeted siRNA could form the basis of clinical trials for RAS-driven benign tumours.

3.
Food Chem Toxicol ; 115: 136-147, 2018 May.
Article in English | MEDLINE | ID: mdl-29518434

ABSTRACT

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is a known human carcinogen. In non-smoking adults greater than 95% of BaP exposure is through diet. The carcinogenicity of BaP is utilized by the U.S. EPA to assess relative potency of complex PAH mixtures. PAH relative potency factors (RPFs, BaP = 1) are determined from high dose animal data. We employed accelerator mass spectrometry (AMS) to determine pharmacokinetics of [14C]-BaP in humans following dosing with 46 ng (an order of magnitude lower than human dietary daily exposure and million-fold lower than animal cancer models). To assess the impact of co-administration of food with a complex PAH mixture, humans were dosed with 46 ng of [14C]-BaP with or without smoked salmon. Subjects were asked to avoid high BaP-containing diets and a 3-day dietary questionnaire given to assess dietary exposure prior to dosing and three days post-dosing with [14C]-BaP. Co-administration of smoked salmon, containing a complex mixture of PAHs with an RPF of 460 ng BaPeq, reduced and delayed absorption. Administration of canned commercial salmon, containing very low amounts of PAHs, showed the impacts on pharmacokinetics were not due to high amounts of PAHs but rather a food matrix effect.


Subject(s)
Benzo(a)pyrene/pharmacokinetics , Carcinogens/pharmacokinetics , Fish Products/analysis , Salmon/metabolism , Adult , Aged , Animals , Benzo(a)pyrene/metabolism , Carbon Radioisotopes/analysis , Carcinogens/metabolism , Cooking , Female , Fish Products/adverse effects , Food Safety , Humans , Male , Middle Aged , Polycyclic Aromatic Hydrocarbons/metabolism , Polycyclic Aromatic Hydrocarbons/pharmacokinetics , Young Adult
4.
J Clin Invest ; 128(4): 1496-1508, 2018 04 02.
Article in English | MEDLINE | ID: mdl-29461977

ABSTRACT

BACKGROUND: Sporadic vascular malformations (VMs) are complex congenital anomalies of blood vessels that lead to stroke, life-threatening bleeds, disfigurement, overgrowth, and/or pain. Therapeutic options are severely limited, and multidisciplinary management remains challenging, particularly for high-flow arteriovenous malformations (AVM). METHODS: To investigate the pathogenesis of sporadic intracranial and extracranial VMs in 160 children in which known genetic causes had been excluded, we sequenced DNA from affected tissue and optimized analysis for detection of low mutant allele frequency. RESULTS: We discovered multiple mosaic-activating variants in 4 genes of the RAS/MAPK pathway, KRAS, NRAS, BRAF, and MAP2K1, a pathway commonly activated in cancer and responsible for the germline RAS-opathies. These variants were more frequent in high-flow than low-flow VMs. In vitro characterization and 2 transgenic zebrafish AVM models that recapitulated the human phenotype validated the pathogenesis of the mutant alleles. Importantly, treatment of AVM-BRAF mutant zebrafish with the BRAF inhibitor vemurafinib restored blood flow in AVM. CONCLUSION: Our findings uncover a major cause of sporadic VMs of different clinical types and thereby offer the potential of personalized medical treatment by repurposing existing licensed cancer therapies. FUNDING: This work was funded or supported by grants from the AVM Butterfly Charity, the Wellcome Trust (UK), the Medical Research Council (UK), the UK National Institute for Health Research, the L'Oreal-Melanoma Research Alliance, the European Research Council, and the National Human Genome Research Institute (US).


Subject(s)
Alleles , MAP Kinase Kinase 1 , MAP Kinase Signaling System/genetics , Mutation , Phenotype , Vascular Malformations , ras Proteins , Adolescent , Adult , Animals , Child , Female , HEK293 Cells , Human Umbilical Vein Endothelial Cells , Humans , Infant , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 1/metabolism , Male , Vascular Malformations/genetics , Vascular Malformations/metabolism , Vascular Malformations/pathology , Zebrafish , ras Proteins/genetics , ras Proteins/metabolism
5.
J Clin Endocrinol Metab ; 103(3): 917-925, 2018 03 01.
Article in English | MEDLINE | ID: mdl-29342293

ABSTRACT

Context: Small for gestational age (SGA) can be the result of fetal growth restriction, which is associated with perinatal morbidity and mortality. Mechanisms that control prenatal growth are poorly understood. Objective: The aim of the current study was to gain more insight into prenatal growth failure and determine an effective diagnostic approach in SGA newborns. We hypothesized that one or more copy number variations (CNVs) and disturbed methylation and sequence variants may be present in genes associated with fetal growth. Design: A prospective cohort study of subjects with a low birth weight for gestational age. Setting: The study was conducted at an academic pediatric research institute. Patients: A total of 21 SGA newborns with a mean birth weight below the first centile and a control cohort of 24 appropriate-for-gestational-age newborns were studied. Interventions: Array comparative genomic hybridization, genome-wide methylation studies, and exome sequencing were performed. Main Outcome Measures: The numbers of CNVs, methylation disturbances, and sequence variants. Results: The genetic analyses demonstrated three CNVs, one systematically disturbed methylation pattern, and one sequence variant explaining SGA. Additional methylation disturbances and sequence variants were present in 20 patients. In 19 patients, multiple abnormalities were found. Conclusion: Our results confirm the influence of a large number of mechanisms explaining dysregulation of fetal growth. We concluded that CNVs, methylation disturbances, and sequence variants all contribute to prenatal growth failure. These genetic workups can be an effective diagnostic approach in SGA newborns.


Subject(s)
Birth Weight/genetics , Fetal Growth Retardation/genetics , Infant, Small for Gestational Age , Comparative Genomic Hybridization , DNA Copy Number Variations , DNA Methylation , Female , Genetic Predisposition to Disease , Genome-Wide Association Study , Gestational Age , Humans , Infant, Newborn , Male , Prospective Studies , Exome Sequencing/methods
6.
Biol Open ; 6(2): 223-231, 2017 Feb 15.
Article in English | MEDLINE | ID: mdl-28069589

ABSTRACT

Syndromic craniosynostosis caused by mutations in FGFR2 is characterised by developmental pathology in both endochondral and membranous skeletogenesis. Detailed phenotypic characterisation of features in the membranous calvarium, the endochondral cranial base and other structures in the axial and appendicular skeleton has not been performed at embryonic stages. We investigated bone development in the Crouzon mouse model (Fgfr2C342Y) at pre- and post-ossification stages to improve understanding of the underlying pathogenesis. Phenotypic analysis was performed by whole-mount skeletal staining (Alcian Blue/Alizarin Red) and histological staining of sections of CD1 wild-type (WT), Fgfr2C342Y/+ heterozygous (HET) and Fgfr2C342Y/C342Y homozygous (HOM) mouse embryos from embryonic day (E)12.5-E17.5 stages. Gene expression (Sox9, Shh, Fgf10 and Runx2) was studied by in situ hybridisation and protein expression (COL2A1) by immunohistochemistry. Our analysis has identified severely decreased osteogenesis in parts of the craniofacial skeleton together with increased chondrogenesis in parts of the endochondral and cartilaginous skeleton in HOM embryos. The Sox9 expression domain in tracheal and basi-cranial chondrocytic precursors at E13.5 in HOM embryos is increased and expanded, correlating with the phenotypic observations which suggest FGFR2 signalling regulates Sox9 expression. Combined with abnormal staining of type II collagen in pre-chondrocytic mesenchyme, this is indicative of a mesenchymal condensation defect. An expanded spectrum of phenotypic features observed in the Fgfr2C342Y/C342Y mouse embryo paves the way towards better understanding the clinical attributes of human Crouzon-Pfeiffer syndrome. FGFR2 mutation results in impaired skeletogenesis; however, our findings suggest that many phenotypic aberrations stem from a primary failure of pre-chondrogenic/osteogenic mesenchymal condensation and link FGFR2 to SOX9, a principal regulator of skeletogenesis.

7.
Mol Carcinog ; 56(1): 163-171, 2017 01.
Article in English | MEDLINE | ID: mdl-26990437

ABSTRACT

The cytochrome P450 (CYP) 1 family is active toward numerous environmental pollutants, including polycyclic aromatic hydrocarbons (PAHs). Utilizing a mouse model, null for Cyp1b1 and expressing human CYP1B1, we tested the hypothesis that hCYP1B1 is important for dibenzo[def,p]chrysene (DBC) transplacental carcinogenesis. Wild-type mCyp1b1, transgenic hCYP1B1 (mCyp1b1 null background), and mCyp1b1 null mice were assessed. Each litter had an equal number of siblings with Ahrb-1/d and Ahrd/d alleles. Pregnant mice were dosed (gavage) on gestation day 17 with 6.5 or 12 mg/kg of DBC or corn oil. At 10 months of age, mortality, general health, lymphoid disease and lung tumor incidence, and multiplicity were assessed. hCYP1B1 genotype did not impact lung tumor multiplicity, but tended to enhance incidence compared to Cyp1b1 wild-type mice (P = 0.07). As with Cyp1b1 in wild-type mice, constitutive hCYP1B1 protein is non-detectable in liver but was induced with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Wild-type mice were 59% more likely to succumb to T-cell Acute Lymphoblastic Leukemia (T-ALL). Unlike an earlier examination of the Ahr genotype in this model (Yu et al., Cancer Res, 2006;66:755-762), but in agreement with a more recent study (Shorey et al., Toxicol Appl Pharmacol, 2013;270:60-69), this genotype was not associated with lung tumor incidence, multiplicity, or mortality. Sex was not significant with respect to lung tumor incidence or mortality but males exhibited significantly greater multiplicity. Lung tumor incidence was greater in mCyp1b1 nulls compared to wild-type mice. To our knowledge, this is the first application of a humanized mouse model in transplacental carcinogenesis. © 2016 Wiley Periodicals, Inc.


Subject(s)
Carcinogenesis/genetics , Cytochrome P-450 CYP1B1/genetics , Lung Neoplasms/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Pregnancy Complications, Neoplastic/genetics , Animals , Carcinogenesis/chemically induced , Carcinogenesis/pathology , Carcinogens , Chrysenes , Female , Gene Expression Regulation, Neoplastic , Humans , Lung/pathology , Lung Neoplasms/chemically induced , Lung Neoplasms/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Placenta/pathology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/chemically induced , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Pregnancy , Pregnancy Complications, Neoplastic/chemically induced , Pregnancy Complications, Neoplastic/pathology
8.
Chem Res Toxicol ; 29(10): 1641-1650, 2016 10 17.
Article in English | MEDLINE | ID: mdl-27494294

ABSTRACT

Metabolism is a key health risk factor following exposures to pro-carcinogenic polycyclic aromatic hydrocarbons (PAHs) such as dibenzo[def,p]chrysene (DBC), an IARC classified 2A probable human carcinogen. Human exposure to PAHs occurs primarily from the diet in nonsmokers. However, little data is available on the metabolism and pharmacokinetics in humans of high molecular weight PAHs (≥4 aromatic rings), including DBC. We previously determined the pharmacokinetics of DBC in human volunteers orally administered a microdose (29 ng; 5 nCi) of [14C]-DBC by accelerator mass spectrometry (AMS) analysis of total [14C] in plasma and urine. In the current study, we utilized a novel "moving wire" interface between ultraperformance liquid chromatography (UPLC) and AMS to detect and quantify parent DBC and its major metabolites. The major [14C] product identified in plasma was unmetabolized [14C]-DBC itself (Cmax = 18.5 ±15.9 fg/mL, Tmax= 2.1 ± 1.0 h), whereas the major metabolite was identified as [14C]-(+/-)-DBC-11,12-diol (Cmax= 2.5 ±1.3 fg/mL, Tmax= 1.8 h). Several minor species of [14C]-DBC metabolites were also detected for which no reference standards were available. Free and conjugated metabolites were detected in urine with [14C]-(+/-)-DBC-11,12,13,14-tetraol isomers identified as the major metabolites, 56.3% of which were conjugated (Cmax= 35.8 ± 23.0 pg/pool, Tmax = 6-12 h pool). [14C]-DBC-11,12-diol, of which 97.5% was conjugated, was also identified in urine (Cmax = 29.4 ± 11.6 pg/pool, Tmax = 6-12 h pool). Parent [14C]-DBC was not detected in urine. This is the first data set to assess metabolite profiles and associated pharmacokinetics of a carcinogenic PAH in human volunteers at an environmentally relevant dose, providing the data necessary for translation of high dose animal models to humans for translation of environmental health risk assessment.


Subject(s)
Benzopyrenes/metabolism , Benzopyrenes/pharmacokinetics , Adult , Aged , Benzopyrenes/analysis , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Female , Healthy Volunteers , Humans , Male , Mass Spectrometry , Middle Aged , Molecular Structure , Young Adult
9.
Comput J ; 58(6): 1431-1442, 2015 Jun 01.
Article in English | MEDLINE | ID: mdl-26146409

ABSTRACT

There is considerable evidence that exposure to air pollution is harmful to health. In the U.S., ambient air quality is monitored by Federal and State agencies for regulatory purposes. There are limited options, however, for people to access this data in real-time which hinders an individual's ability to manage their own risks. This paper describes a new software package that models environmental concentrations of fine particulate matter (PM2.5), coarse particulate matter (PM10), and ozone concentrations for the state of Oregon and calculates personal health risks at the smartphone's current location. Predicted air pollution risk levels can be displayed on mobile devices as interactive maps and graphs color-coded to coincide with EPA air quality index (AQI) categories. Users have the option of setting air quality warning levels via color-coded bars and were notified whenever warning levels were exceeded by predicted levels within 10 km. We validated the software using data from participants as well as from simulations which showed that the application was capable of identifying spatial and temporal air quality trends. This unique application provides a potential low-cost technology for reducing personal exposure to air pollution which can improve quality of life particularly for people with health conditions, such as asthma, that make them more susceptible to these hazards.

10.
Toxicol Appl Pharmacol ; 287(2): 149-160, 2015 Sep 01.
Article in English | MEDLINE | ID: mdl-26049101

ABSTRACT

FVB/N mice wild-type, heterozygous or null for Cyp 1b1 were used in a two-stage skin tumor study comparing PAH, benzo[a]pyrene (BaP), dibenzo[def,p]chrysene (DBC), and coal tar extract (CTE, SRM 1597a). Following 20 weeks of promotion with TPA the Cyp 1b1 null mice, initiated with DBC, exhibited reductions in incidence, multiplicity, and progression. None of these effects were observed with BaP or CTE. The mechanism of Cyp 1b1-dependent alteration of DBC skin carcinogenesis was further investigated by determining expression of select genes in skin from DBC-treated mice 2, 4 and 8h post-initiation. A significant reduction in levels of Cyp 1a1, Nqo1 at 8h and Akr 1c14 mRNA was observed in Cyp 1b1 null (but not wt or het) mice, whereas no impact was observed in Gst a1, Nqo 1 at 2 and 4h or Akr 1c19 at any time point. Cyp 1b1 mRNA was not elevated by DBC. The major covalent DNA adducts, dibenzo[def,p]chrysene-(±)-11,12-dihydrodiol-cis and trans-13,14-epoxide-deoxyadenosine (DBCDE-dA) were quantified by UHPLC-MS/MS 8h post-initiation. Loss of Cyp1 b1 expression reduced DBCDE-dA adducts in the skin but not to a statistically significant degree. The ratio of cis- to trans-DBCDE-dA adducts was higher in the skin than other target tissues such as the spleen, lung and liver (oral dosing). These results document that Cyp 1b1 plays a significant role in bioactivation and carcinogenesis of DBC in a two-stage mouse skin tumor model and that loss of Cyp 1b1 has little impact on tumor response with BaP or CTE as initiators.


Subject(s)
Carcinogens/toxicity , Coal Tar/toxicity , Cytochrome P-450 CYP1B1/metabolism , Polycyclic Aromatic Hydrocarbons/toxicity , Skin Neoplasms/chemically induced , Animals , Benzopyrenes , Cytochrome P-450 CYP1B1/genetics , DNA Adducts/metabolism , Female , Gene Expression , Mice , Mice, Knockout , RNA, Messenger/biosynthesis , Tandem Mass Spectrometry , Time Factors
11.
Toxicol Sci ; 146(1): 135-45, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25908611

ABSTRACT

We have previously shown that relative potency factors and DNA adduct measurements are inadequate for predicting carcinogenicity of certain polycyclic aromatic hydrocarbons (PAHs) and PAH mixtures, particularly those that function through alternate pathways or exhibit greater promotional activity compared to benzo[a]pyrene (BaP). Therefore, we developed a pathway-based approach for classification of tumor outcome after dermal exposure to PAH/mixtures. FVB/N mice were exposed to dibenzo[def,p]chrysene (DBC), BaP, or environmental PAH mixtures (Mix 1-3) following a 2-stage initiation/promotion skin tumor protocol. Resulting tumor incidence could be categorized by carcinogenic potency as DBC >> BaP = Mix2 = Mix3 > Mix1 = Control, based on statistical significance. Gene expression profiles measured in skin of mice collected 12 h post-initiation were compared with tumor outcome for identification of short-term bioactivity profiles. A Bayesian integration model was utilized to identify biological pathways predictive of PAH carcinogenic potential during initiation. Integration of probability matrices from four enriched pathways (P < .05) for DNA damage, apoptosis, response to chemical stimulus, and interferon gamma signaling resulted in the highest classification accuracy with leave-one-out cross validation. This pathway-driven approach was successfully utilized to distinguish early regulatory events during initiation prognostic for tumor outcome and provides proof-of-concept for using short-term initiation studies to classify carcinogenic potential of environmental PAH mixtures. These data further provide a 'source-to-outcome' model that could be used to predict PAH interactions during tumorigenesis and provide an example of how mode-of-action-based risk assessment could be employed for environmental PAH mixtures.


Subject(s)
Carcinogens/classification , Carcinogens/toxicity , Polycyclic Aromatic Hydrocarbons/classification , Polycyclic Aromatic Hydrocarbons/toxicity , Animals , Mice
12.
PLoS One ; 8(11): e79153, 2013.
Article in English | MEDLINE | ID: mdl-24236101

ABSTRACT

BACKGROUND: One of the rationales behind using strength training in the treatment of adolescents with Patellofemoral Pain (PFP) is that reduced strength of the lower extremity is a risk factor for PFP and a common deficit. This rationale is based on research conducted on adolescents >15 years of age but has never been investigated among young adolescents with PFP. OBJECTIVES: To compare isometric muscle strength of the lower extremity among adolescents with PFP compared to age- and gender-matched pain-free adolescents. METHODS: In 2011 a population-based cohort (APA2011-cohort) consisting of 768 adolescents aged 12-15 years from 8 local schools was formed. In September 2012, all adolescents who reported knee pain in September 2011 were offered a clinical examination if they still had knee pain. From these, 20 adolescents (16 females) were diagnosed with PFP. Pain-free adolescents from the APA2011-cohort (n = 20) were recruited on random basis as age- and gender-matched pairs. Primary outcome was isometric knee extension strength normalized to body weight (%BW) and blinded towards subject information. Secondary outcomes included knee flexion, hip abduction/adduction and hip internal/external rotation strength. Demographic data included Knee Injury and Osteoarthritis Outcome Score (KOOS) and symptom duration. RESULTS: Adolescents with PFP reported long symptom duration and significantly worse KOOS scores compared to pain-free adolescents. There were no significant differences in isometric knee extension strength (Δ0.3% BW, p = 0.97), isometric knee flexion strength (Δ0.4% BW, p = 0.84) or different measures of hip strength (Δ0.4 to 1.1% BW, p>0.35). CONCLUSION: Young symptomatic adolescents with PFP between 12 and 16 years of age did not have decreased isometric muscle strength of the knee and hip. These results question the rationale of targeting strength deficits in the treatment of adolescents with PFP. However, strength training may still be an effective treatment for those individuals with PFP suffering from strength deficits.


Subject(s)
Hip/physiopathology , Knee/physiopathology , Patellofemoral Pain Syndrome/physiopathology , Adolescent , Case-Control Studies , Child , Cross-Sectional Studies , Female , Humans , Male , Muscle Strength
13.
Aviat Space Environ Med ; 84(11): 1153-8, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24279228

ABSTRACT

PURPOSE: This study examined the influence of body fatness on body core temperature and heat loss responses during moderate-intensity exercise. METHODS: Nine men with lower body fat and eight men with higher body fat, matched for aerobic fitness, completed 1 h of recumbent cycling at the same absolute intensity in a warm environment (30 degrees C, 40% RH). Percent body fat was measured by hydrostatic weighing, using oxygen dilution to determine residual volume. Esophageal temperature (T(es)), mean skin temperature (T(sk)), and local sweat rate (m(sw)) were measured at rest and continuously during exercise while forearm blood flow (FBF) was measured at rest and every 10 min during exercise. RESULTS: The lower body fat and higher body fat groups were successfully matched for aerobic fitness, removing the influence of body fatness, given that V/O2(peak) was 50.72 +/- 7.34 and 50.43 +/- 5.01 ml x kg LBM(-1) x min(-1), respectively. When compared to lower body fat individuals, % body fat, body surface area (A(D)), and body mass were higher and A(D)/ mass was lower in higher body fat individuals. T(es), T(sk), FBF, m(sw), and the slope of m(sw):T(es) were not different between groups. Metabolic heat production was similar between the lower body fat (299.7 +/- 40.5 W x m(-2)) and higher body fat (288.1 +/- 30.6 W x m(-2)) subjects, respectively. Dry and evaporative heat loss, as well as heat storage during exercise, were not different between groups. CONCLUSION: These data suggest that there is no effect of body fatness on body core temperature or heat loss responses during moderate-intensity exercise in a warm environment.


Subject(s)
Adiposity/physiology , Body Temperature Regulation/physiology , Body Temperature/physiology , Exercise/physiology , Adult , Female , Heat Stress Disorders/physiopathology , Humans , Male , Oxygen Consumption/physiology , Young Adult
14.
Toxicol Sci ; 135(1): 48-62, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23744095

ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants generated during combustion. Dibenzo[def,p]chrysene (DBC) is a high molecular weight PAH classified as a 2B carcinogen by the International Agency for Research on Cancer. DBC crosses the placenta in exposed mice, causing carcinogenicity in offspring. We present pharmacokinetic data of DBC in pregnant and nonpregnant mice. Pregnant (gestational day 17) and nonpregnant female B6129SF1/J mice were exposed to 15mg/kg DBC by oral gavage. Subgroups of mice were sacrificed up to 48h postdosing, and blood, excreta, and tissues were analyzed for DBC and its major diol and tetrol metabolites. Elevated maximum concentrations and areas under the curve of DBC and its metabolites were observed in blood and tissues of pregnant animals compared with naïve mice. Using a physiologically based pharmacokinetic (PBPK) model, we found observed differences in pharmacokinetics could not be attributed solely to changes in tissue volumes and blood flows that occur during pregnancy. Measurement of enzyme activity in naïve and pregnant mice by activity-based protein profiling indicated a 2- to 10-fold reduction in activities of many of the enzymes relevant to PAH metabolism. Incorporating this reduction into the PBPK model improved model predictions. Concentrations of DBC in fetuses were one to two orders of magnitude below maternal blood concentrations, whereas metabolite concentrations closely resembled those observed in maternal blood.


Subject(s)
Benzopyrenes/pharmacokinetics , Carcinogens/pharmacokinetics , Pregnancy, Animal/metabolism , Animals , Aryl Hydrocarbon Hydroxylases/physiology , Cytochrome P-450 CYP1B1 , Female , Male , Mice , Models, Biological , Pregnancy , Tissue Distribution
15.
Biomacromolecules ; 14(6): 1850-8, 2013 Jun 10.
Article in English | MEDLINE | ID: mdl-23617462

ABSTRACT

The competitive adsorption between whey protein concentrate (WPC) or sodium caseinate (SCN) and four bile salts, sodium cholate (NaC), dexocycholate (NaDC), taurocholate (NaTC), and glycodeoxycholate (NaGDC), has been studied in protein stabilized oil-in-water emulsions. The bile salts that contain a conjugated amino acid (NaTC and NaGDC) were considerably more efficient at displacing both WPC and SCN proteins from the emulsion droplet interface, even though they are known to have a hydrophobicity lower than that of NaC and NaDC. This is explained in terms of a steric resistance to adsorption from the conjugated amino acids in NaTC and NaGDC. This leads to their adopting an adsorbed conformation at the oil-water interface that penetrates less into the oil phase, causing greater disruption of the adsorbed layer, and hence leads to greater displacement of protein from the interface. Complementary computer simulations of the adsorption of the four bile salts at the decane-water interface support the hypothesis that the NaTC and NaGDC adopt flatter conformations that stick out further into the aqueous phase, which arises from a lower free energy of adsorption. The surface coverage as a function of bulk concentration for the four bile salts has also been measured. These have been found to have a form that fits closely the Langmuir-Freundlich isotherm. The results for NaC suggest that it adsorbs as individual molecules and forms a saturated monolayer over much of the concentration range used in the displacement experiments, since it is below its critical micelle concentration in this range. For the other three bile salts, on the other hand, the primary adsorbing species appears to be the micelle form, since the surface coverage is above that of a saturated monolayer for much of the concentration range studied.


Subject(s)
Bile Acids and Salts/chemistry , Caseins/chemistry , Emulsions , Milk Proteins/chemistry , Oils , Water , Adsorption , Molecular Dynamics Simulation , Whey Proteins
16.
Mol Ther ; 21(5): 1014-23, 2013 May.
Article in English | MEDLINE | ID: mdl-23481323

ABSTRACT

Understanding the host response to oncolytic viruses is important to maximize their antitumor efficacy. Despite robust cytotoxicity and high virus production of an oncolytic herpes simplex virus (oHSV) in cultured human sarcoma cells, intratumoral (ITu) virus injection resulted in only mild antitumor effects in some xenograft models, prompting us to characterize the host inflammatory response. Virotherapy induced an acute neutrophilic infiltrate, a relative decrease of ITu macrophages, and a myeloid cell-dependent upregulation of host-derived vascular endothelial growth factor (VEGF). Anti-VEGF antibodies, bevacizumab and r84, the latter of which binds VEGF and selectively inhibits binding to VEGF receptor-2 (VEGFR2) but not VEGFR1, enhanced the antitumor effects of virotherapy, in part due to decreased angiogenesis but not increased virus production. Neither antibody affected neutrophilic infiltration but both partially mitigated virus-induced depletion of macrophages. Enhancement of virotherapy-mediated antitumor effects by anti-VEGF antibodies could largely be recapitulated by systemic depletion of CD11b(+) cells. These data suggest the combined effect of oHSV virotherapy and anti-VEGF antibodies is in part due to modulation of a host inflammatory reaction to virus. Our data provide strong preclinical support for combined oHSV and anti-VEGF antibody therapy and suggest that understanding and counteracting the innate host response may help enable the full antitumor potential of oncolytic virotherapy.


Subject(s)
Genetic Vectors/immunology , Myeloid Cells/immunology , Neoplasms/immunology , Oncolytic Viruses/immunology , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Animals , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal, Humanized/pharmacology , Bevacizumab , CD11b Antigen/metabolism , Cell Culture Techniques , Cell Line, Tumor , Disease Models, Animal , Female , Genetic Vectors/administration & dosage , Humans , Macrophages/immunology , Macrophages/metabolism , Mice , Myeloid Cells/metabolism , Neoplasms/metabolism , Neoplasms/therapy , Neovascularization, Pathologic/therapy , Oncolytic Virotherapy , Sarcoma/immunology , Sarcoma/metabolism , Sarcoma/therapy , Simplexvirus/immunology , Stromal Cells/metabolism , Stromal Cells/virology , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/immunology , Virus Replication/drug effects , Xenograft Model Antitumor Assays
17.
Toxicol Appl Pharmacol ; 267(2): 192-9, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23274566

ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs) are present in the environment as complex mixtures with components that have diverse carcinogenic potencies and mostly unknown interactive effects. Non-additive PAH interactions have been observed in regulation of cytochrome P450 (CYP) gene expression in the CYP1 family. To better understand and predict biological effects of complex mixtures, such as environmental PAHs, an 11 gene input-1 gene output fuzzy neural network (FNN) was developed for predicting PAH-mediated perturbations of dermal Cyp1b1 transcription in mice. Input values were generalized using fuzzy logic into low, medium, and high fuzzy subsets, and sorted using k-means clustering to create Mamdani logic functions for predicting Cyp1b1 mRNA expression. Model testing was performed with data from microarray analysis of skin samples from FVB/N mice treated with toluene (vehicle control), dibenzo[def,p]chrysene (DBC), benzo[a]pyrene (BaP), or 1 of 3 combinations of diesel particulate extract (DPE), coal tar extract (CTE) and cigarette smoke condensate (CSC) using leave-one-out cross-validation. Predictions were within 1 log(2) fold change unit of microarray data, with the exception of the DBC treatment group, where the unexpected down-regulation of Cyp1b1 expression was predicted but did not reach statistical significance on the microarrays. Adding CTE to DPE was predicted to increase Cyp1b1 expression, whereas adding CSC to CTE and DPE was predicted to have no effect, in agreement with microarray results. The aryl hydrocarbon receptor repressor (Ahrr) was determined to be the most significant input variable for model predictions using back-propagation and normalization of FNN weights.


Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Fuzzy Logic , Gene Regulatory Networks/drug effects , Neural Networks, Computer , Polycyclic Aromatic Hydrocarbons/toxicity , Skin/drug effects , Animals , Cytochrome P-450 CYP1B1 , Female , Mice , Risk Assessment
18.
Toxicol Appl Pharmacol ; 264(3): 377-86, 2012 Nov 01.
Article in English | MEDLINE | ID: mdl-22935520

ABSTRACT

The polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), was compared to dibenzo[def,p]chrysene (DBC) and combinations of three environmental PAH mixtures (coal tar, diesel particulate and cigarette smoke condensate) using a two stage, FVB/N mouse skin tumor model. DBC (4nmol) was most potent, reaching 100% tumor incidence with a shorter latency to tumor formation, less than 20 weeks of 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion compared to all other treatments. Multiplicity was 4 times greater than BaP (400 nmol). Both PAHs produced primarily papillomas followed by squamous cell carcinoma and carcinoma in situ. Diesel particulate extract (1 mg SRM 1650b; mix 1) did not differ from toluene controls and failed to elicit a carcinogenic response. Addition of coal tar extract (1 mg SRM 1597a; mix 2) produced a response similar to BaP. Further addition of 2 mg of cigarette smoke condensate (mix 3) did not alter the response with mix 2. PAH-DNA adducts measured in epidermis 12 h post initiation and analyzed by ³²P post-labeling, did not correlate with tumor incidence. PAH-dependent alteration in transcriptome of skin 12 h post initiation was assessed by microarray. Principal component analysis (sum of all treatments) of the 922 significantly altered genes (p<0.05), showed DBC and BaP to cluster distinct from PAH mixtures and each other. BaP and mixtures up-regulated phase 1 and phase 2 metabolizing enzymes while DBC did not. The carcinogenicity with DBC and two of the mixtures was much greater than would be predicted based on published Relative Potency Factors (RPFs).


Subject(s)
Benzo(a)pyrene/toxicity , Benzopyrenes/toxicity , Carcinogens, Environmental/toxicity , Skin Neoplasms/chemically induced , Animals , Benzo(a)pyrene/metabolism , Benzopyrenes/metabolism , Carcinogens, Environmental/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Mice , Mice, Inbred Strains , Molecular Structure , Principal Component Analysis , Protein Array Analysis , Skin Neoplasms/metabolism , Transcriptome
19.
Cancer Lett ; 317(1): 49-55, 2012 Apr 01.
Article in English | MEDLINE | ID: mdl-22085489

ABSTRACT

Dibenzo[def,p]chrysene (DBC) is a transplacental carcinogen in mice (15mg/kg; gestation day (GD) 17). To mimic residual exposure throughout pregnancy, dams received four smaller doses of DBC (3.75mg/kg) on GD 5, 9, 13 and 17. This regimen alleviated the previously established carcinogenic responses in the thymus, lung, and liver. However, there was a marked increase in ovarian tumors (females) and hyperplastic testes (males). [(14)C]-DBC (GD 17) dosing revealed transplacental distribution to fetal tissues at 10-fold lower concentrations than in paired maternal tissue and residual [(14)C] 3weeks post-dose. This study highlights the importance of developmental stage in susceptibility to environmental carcinogens.


Subject(s)
Benzopyrenes/toxicity , Carcinogens/toxicity , Maternal Exposure , Maternal-Fetal Exchange , Neoplasms, Experimental/chemically induced , Placental Circulation , Prenatal Exposure Delayed Effects , Animals , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , Benzopyrenes/administration & dosage , Benzopyrenes/pharmacokinetics , Carcinogens/administration & dosage , Carcinogens/pharmacokinetics , Cytochrome P-450 CYP1B1 , Female , Fetus/drug effects , Fetus/metabolism , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Gestational Age , Male , Mice , Mice, 129 Strain , Neoplasms, Experimental/pathology , Pregnancy , Time Factors , Tissue Distribution
20.
Mutat Res ; 714(1-2): 53-62, 2011 Sep 01.
Article in English | MEDLINE | ID: mdl-21762708

ABSTRACT

Many of the toxic and carcinogenic effects of urban air pollution have been linked to polycyclic aromatic hydrocarbons (PAHs) adsorbed to airborne particulate matter (PM). The carcinogenic properties of PAHs in complex organic mixtures derived from PM have been chiefly attributed to their mutagenicity. Nevertheless, PAHs are also potent activators of the aryl hydrocarbon receptor (AhR), which may contribute to their nongenotoxic effects, including tumor promotion. As the genotoxicity of carcinogenic PAHs in complex mixtures derived from urban PM is often inhibited by other mixture constituents, the AhR-mediated activity of urban PM extracts might significantly contribute to the carcinogenic activity of such mixtures. In the present study, we used an organic extract of the urban dust standard reference material, SRM1649a, as a model mixture to study a range of toxic effects related to DNA damage and AhR activation. Both the organic extract and its neutral aromatic fraction formed a low number of DNA adducts per nucleotide in the liver epithelial WB-F344 cells model, without inducing DNA damage response, such as tumor suppressor p53 activation and apoptosis. In contrast, we found that this extract, as well as its neutral and polar fractions, were potent inducers of a range of AhR-mediated responses, including induction of the AhR-mediated transcription, such as cytochrome P450 1A1/1B1 expression, and the AhR-dependent cell proliferation. Importantly, these toxic events occurred at doses one order of magnitude lower than DNA damage. The AhR-mediated activity of the neutral fraction was linked to PAHs and their derivatives, as polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls were only minor contributors to the overall AhR-mediated activity. Taken together, our data suggest that more attention should be paid to the AhR-dependent nongenotoxic events elicited by urban PM constituents, especially PAHs and their derivatives.


Subject(s)
DNA Damage/drug effects , Mutagens/toxicity , Organic Chemicals/toxicity , Particulate Matter/toxicity , Polycyclic Aromatic Hydrocarbons/toxicity , Receptors, Aryl Hydrocarbon/metabolism , Animals , Apoptosis/drug effects , Cell Line , Cell Proliferation/drug effects , Cytochrome P-450 CYP1A1/metabolism , DNA Adducts/drug effects , Dose-Response Relationship, Drug , Genes, p53/drug effects , Liver/drug effects , Rats
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