Toxicokinetics of benzo[a]pyrene in humans: Extensive metabolism as determined by UPLC-accelerator mass spectrometry following oral micro-dosing.

Benzo[a]pyrene (BaP), is a known human carcinogen (International Agency for Research on Cancer (IARC) class 1). The remarkable sensitivity (zepto-attomole 14C in biological samples) of accelerator mass spectrometry (AMS) makes possible, with de minimus risk, pharmacokinetic (PK) analysis following [14C]-BaP micro-dosing of humans. A 46 ng (5 nCi) dose was given thrice to 5 volunteers with minimum 2 weeks between dosing and plasma collected over 72 h. [14C]-BaPeq PK analysis gave plasma Tmax and Cmax values of 1.25 h and 29-82 fg/mL, respectively. PK parameters were assessed by non- compartment and compartment models. Intervals between dosing ranged from 20 to 420 days and had little impact on intra-individual variation. DNA, extracted from peripheral blood mononuclear cells (PBMCs) of 4 volunteers, showed measurable levels (LOD ~ 0.5 adducts/1011 nucleotides) in two individuals 2-3 h post-dose, approximately three orders of magnitude lower than smokers or occupationally-exposed individuals. Little or no DNA binding was detectable at 48-72 h. In volunteers the allelic variants CYP1B1*1/*⁎1, *1/*3 or *3/*3 and GSTM1*0/0 or *1 had no impact on [14C]-BaPeq PK or DNA adduction with this very limited sample. Plasma metabolites over 72 h from two individuals (one CYP1B1*1/*1 and one CYP1B1*3/*3) were analyzed by UPLC-AMS. In both individuals, parent [14C]-BaP was a minor constituent even at the earliest time points and metabolite profiles markedly distinct. AMS, coupled with UPLC, could be used in humans to enhance the accuracy of pharmacokinetics, toxicokinetics and risk assessment of environmental carcinogens.

Detection of unique Ebola virus oligonucleotides using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide probe pairs.

Here we identify a low-cost diagnostic platform using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide (PMO) probe pairs, which upon binding target oligonucleotides undergo fluorescence resonance energy transfer (FRET). Using a target oligonucleotide derived from the Ebola virus (EBOV), we have derivatized PMO probes with either Alexa Fluor488 (donor) or tetramethylrhodamine (acceptor). Upon EBOV target oligonulceotide binding, observed changes in FRET between PMO probe pairs permit a 25 pM lower limit of detection; there is no off-target binding within a complex mixture of nucleic acids and other biomolecules present in human saliva. Equivalent levels of FRET occur using PMO probe pairs for single or double stranded oligonucleotide targets. High-affinity binding is retained under low-ionic strength conditions that disrupt oligonucleotide secondary structures (e.g., stem-loop structures), ensuring reliable target detection. Under these low-ionic strength conditions, rates of PMO probe binding to target oligonucleotides are increased 3-fold relative to conventional high-ionic strength conditions used for nucleic acid hybridization, with half-maximal binding occurring within 10 min. Our results indicate an ability to use PMO probe pairs to detect clinically relevant levels of EBOV and other oligonucleotide targets in complex biological samples without the need for nucleic acid amplification, and open the possibility of population screening that includes assays for the genomic integration of DNA based copies of viral RNA.

Pharmacokinetics of [14C]-Benzo[a]pyrene (BaP) in humans: Impact of Co-Administration of smoked salmon and BaP dietary restriction.

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.

Human Microdosing with Carcinogenic Polycyclic Aromatic Hydrocarbons: In Vivo Pharmacokinetics of Dibenzo[def,p]chrysene and Metabolites by UPLC Accelerator Mass Spectrometry.

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.

Cytochrome P450 1b1 in polycyclic aromatic hydrocarbon (PAH)-induced skin carcinogenesis: Tumorigenicity of individual PAHs and coal-tar extract, DNA adduction and expression of select genes in the Cyp1b1 knockout mouse.

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.

Analysis of dibenzo[def,p]chrysene-deoxyadenosine adducts in wild-type and cytochrome P450 1b1 knockout mice using stable-isotope dilution UHPLC-MS/MS.

The polycyclic aromatic hydrocarbon (PAH), dibenzo[def,p]chrysene (DBC; also known as dibenzo[a,l]pyrene), is a potent carcinogen in animal models and a class 2A human carcinogen. Recent investigations into DBC-mediated toxicity identified DBC as a potent immunosuppressive agent similar to the well-studied immunotoxicant 7,12-dimethylbenz[a]anthracene (DMBA). DBC, like DMBA, is bioactivated by cytochrome P450 (CYP) 1B1 and forms the reactive metabolite DBC-11,12-diol-13,14-epoxide (DBCDE). DBCDE is largely responsible for the genotoxicity associated with DBC exposure. The immunosuppressive properties of several PAHs are also linked to genotoxic mechanisms. Therefore, this study was designed to identify DBCDE-DNA adduct formation in the spleen and thymus of wild-type and cytochrome P450 1b1 (Cyp1b1) knockout (KO) mice using a highly sensitive stable-isotope dilution UHPLC-MS/MS method. Stable-isotope dilution UHPLC-MS/MS identified the major DBC adducts (±)-anti-cis-DBCDE-dA and (±)-anti-trans-DBCDE-dA in the lung, liver, and spleen of both WT and Cyp1b1 KO mice. However, adduct formation in the thymus was below the level of quantitation for our method. Additionally, adduct formation in Cyp1b1 KO mice was significantly reduced compared to wild-type (WT) mice receiving DBC via oral gavage. In conclusion, the current study identifies for the first time DBCDE-dA adducts in the spleen of mice supporting the link between genotoxicity and immunosuppression, in addition to supporting previous studies identifying Cyp1b1 as the primary CYP involved in DBC bioactivation to DBCDE. The high levels of DBC-DNA adducts identified in the spleen, along with the known high levels of Cyp1b1 expression in this organ, supports further investigation into DBC-mediated immunotoxicity.

Human in Vivo Pharmacokinetics of [(14)C]Dibenzo[def,p]chrysene by Accelerator Mass Spectrometry Following Oral Microdosing.

Dibenzo(def,p)chrysene (DBC), (also known as dibenzo[a,l]pyrene), is a high molecular weight polycyclic aromatic hydrocarbon (PAH) found in the environment, including food, produced by the incomplete combustion of hydrocarbons. DBC, classified by IARC as a 2A probable human carcinogen, has a relative potency factor (RPF) in animal cancer models 30-fold higher than benzo[a]pyrene. No data are available describing the disposition of high molecular weight (>4 rings) PAHs in humans to compare to animal studies. Pharmacokinetics of DBC was determined in 3 female and 6 male human volunteers following oral microdosing (29 ng, 5 nCi) of [(14)C]-DBC. This study was made possible with highly sensitive accelerator mass spectrometry (AMS), capable of detecting [(14)C]-DBC equivalents in plasma and urine following a dose considered of de minimus risk to human health. Plasma and urine were collected over 72 h. The plasma Cmax was 68.8 ± 44.3 fg·mL(-1) with a Tmax of 2.25 ± 1.04 h. Elimination occurred in two distinct phases: a rapid (α)-phase, with a T1/2 of 5.8 ± 3.4 h and an apparent elimination rate constant (Kel) of 0.17 ± 0.12 fg·h(-1), followed by a slower (ß)-phase, with a T1/2 of 41.3 ± 29.8 h and an apparent Kel of 0.03 ± 0.02 fg·h(-1). In spite of the high degree of hydrophobicity (log Kow of 7.4), DBC was eliminated rapidly in humans, as are most PAHs in animals, compared to other hydrophobic persistent organic pollutants such as, DDT, PCBs and TCDD. Preliminary examination utilizing a new UHPLC-AMS interface, suggests the presence of polar metabolites in plasma as early as 45 min following dosing. This is the first in vivo data set describing pharmacokinetics in humans of a high molecular weight PAH and should be a valuable addition to risk assessment paradigms.

Cancer chemoprevention by dietary chlorophylls: a 12,000-animal dose-dose matrix biomarker and tumor study.

Recent pilot studies found natural chlorophyll (Chl) to inhibit carcinogen uptake and tumorigenesis in rodent and fish models, and to alter uptake and biodistribution of trace (14)C-aflatoxin B1 in human volunteers. The present study extends these promising findings, using a dose-dose matrix design to examine Chl-mediated effects on dibenzo(def,p)chrysene (DBC)-induced DNA adduct formation, tumor incidence, tumor multiplicity, and changes in gene regulation in the trout. The dose-dose matrix design employed an initial 12,360 rainbow trout, which were treated with 0-4000ppm dietary Chl along with 0-225ppm DBC for up to 4weeks. Dietary DBC was found to induce dose-responsive changes in gene expression that were abolished by Chl co-treatment, whereas Chl alone had no effect on the same genes. Chl co-treatment provided a dose-responsive reduction in total DBC-DNA adducts without altering relative adduct intensities along the chromatographic profile. In animals receiving DBC alone, liver tumor incidence (as logit) and tumor multiplicity were linear in DBC dose (as log) up to their maximum-effect dose, and declined thereafter. Chl co-treatment substantially inhibited incidence and multiplicity at DBC doses up to their maximum-effect dose. These results show that Chl concentrations encountered in Chl-rich green vegetables can provide substantial cancer chemoprotection, and suggest that they do so by reducing carcinogen bioavailability. However, at DBC doses above the optima, Chl co-treatments failed to inhibit tumor incidence and significantly enhanced multiplicity. This finding questions the human relevance of chemoprevention studies carried out at high carcinogen doses that are not proven to lie within a linear, or at least monotonic, endpoint dose-response range.

Low-dose dietary chlorophyll inhibits multi-organ carcinogenesis in the rainbow trout.

We recently reported that chlorophyll (Chl) strongly inhibits aflatoxin B(1) preneoplasia biomarkers in rats when administered by co-gavage (Simonich et al., 2007. Natural chlorophyll inhibits aflatoxin B1-induced multi-organ carcinogenesis in the rat. Carcinogenesis 28, 1294-1302.). The present study extends this by examining the effects of dietary Chl on tumor development, using rainbow trout to explore ubiquity of mechanism. Duplicate groups of 140 trout were fed diet containing 224 ppm dibenzo[a,l]pyrene (DBP) alone, or with 1000-6000 ppm Chl, for 4 weeks. DBP induced high tumor incidences in liver (51%) and stomach (56%), whereas Chl co-fed at 2000, 4000 or 6000 ppm reduced incidences in stomach (to 29%, 23% and 19%, resp., P<0.005) and liver (to 21%, 28% and 26%, resp., P<0.0005). Chlorophyllin (CHL) at 2000 ppm gave similar protection. Chl complexed with DBP in vitro (2Chl:DBP, K(d1)=4.44+/-0.46 microM, K(d2)=3.30+/-0.18 microM), as did CHL (K(d1)=1.38+/-0.32 microM, K(d2)=1.17+/-0.05 microM), possibly explaining their ability to inhibit DBP uptake into the liver by 61-63% (P<0.001). This is the first demonstration that dietary Chl can reduce tumorigenesis in any whole animal model, and that it may do so by a simple, species-independent mechanism.

Protective effect of dietary tomatine against dibenzo[a,l]pyrene (DBP)-induced liver and stomach tumors in rainbow trout.

The potential anti-carcinogenic effects of tomatine, a mixture of commercial tomato glycoalkaloids alpha-tomatine and dehydrotomatine (10:1), were examined in the rainbow trout chemoprevention model. Prior to the chemoprevention study, a preliminary toxicity study revealed that tomatine in the diet fed daily at doses from 100 to 2000 parts per million (ppm) for 4 weeks was not toxic to trout. For the tumor study, replicate groups of 105 trout were fed diets containing dibenzo[a,l]pyrene (DBP) alone (224 ppm), (N = 3), DBP plus tomatine at 2000 ppm (N = 2), tomatine alone (N = 2), or control diet (N = 2) for 4 weeks. The fish were then returned to control diet for 8 months and necropsied for histopathology. Dietary tomatine was found to reduce DBP-initiated liver tumor incidence from 37.0 to 19.0% and stomach tumor incidence from 46.4 to 29.4%. Tomatine also reduced stomach tumor multiplicity. The tomatine-containing diets did not induce mortality, change in fish weights, or liver weights. No adverse pathological effects in the tissues of the fish on the tomatine diets were observed. Dose-response and chemopreventive mechanisms for tomatine protection remain to be examined. This is the first report on the anticarcinogenic effects of tomatine in vivo.

Spatial regulation of Galphai protein signaling in clathrin-coated membrane microdomains containing GAIP.

Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that bind to Galpha subunits and attenuate G protein signaling, but where these events occur in the cell is not yet established. Here we investigated, by immunofluorescence labeling and deconvolution analysis, the site at which endogenous Galpha-interacting protein (GAIP) (RGS19) binds to Galphai3-YFP and its fate after activation of delta-opioid receptor (DOR). In the absence of agonist, GAIP is spatially segregated from Galphai3 and DOR in clathrin-coated domains (CCPs) of the cell membrane (PM), whereas Galphai3-YPF and DOR are located in non-clathrin-coated microdomains of the PM. Upon addition of agonist, Galphai3 partially colocalizes with GAIP in CCPs at the PM. When endocytosis is blocked by expression of a dynamin mutant [dyn(K44A)], there is a striking overlap in the distribution of DOR and Galphai3-YFP with GAIP in CCPs. Moreover, Galphai3-YFP and GAIP form a coprecipitable complex. Our results support a model whereby, after agonist addition, DOR and Galphai3 move together into CCPs where Galphai3 and GAIP meet and turn off G protein signaling. Subsequently, Galphai3 returns to non-clathrin-coated microdomains of the PM, GAIP remains stably associated with CCPs, and DOR is internalized via clathrin-coated vesicles. This constitutes a novel mechanism for regulation of Galpha signaling through spatial segregation of a GAP in clathrin-coated pits.

GAIP, GIPC and Galphai3 are concentrated in endocytic compartments of proximal tubule cells: putative role in regulating megalin's function.

Megalin is the most abundant endocytic receptor in the proximal tubule epithelium (PTE), where it is concentrated in clathrin-coated pits (CCPs) and vesicles in the brush border region. The heterotrimeric G protein alpha subunit, Galphai3, has also been localized to the brush border region of PTE. By immunofluorescence GIPC and GAIP, components of G protein-mediated signaling pathways, are also concentrated in the brush border region of PTE and are present in megalin-expressing cell lines. By cell fractionation, these signaling molecules cosediment with megalin in brush border and microvillar fractions. GAIP is found by immunoelectron microscopy in CCPs, and GIPC is found in CCPs and apical tubules of endocytic compartments in the renal brush border. In precipitation assays, GST-GIPC specifically binds megalin. The concentration of Galphai3, GIPC, and GAIP with megalin in endocytic compartments of the proximal tubule, where extensive endocytosis occurs, and the interaction between GIPC and the cytoplasmic tail of megalin suggest a model whereby G protein-mediated signaling may regulate megalin's endocytic function and/or trafficking.

The glomerular epithelial cell anti-adhesin podocalyxin associates with the actin cytoskeleton through interactions with ezrin.

During development, renal glomerular epithelial cells (podocytes) undergo extensive morphologic changes necessary for creation of the glomerular filtration apparatus. These changes include formation of interdigitating foot processes, replacement of tight junctions with slit diaphragms, and the concomitant opening of intercellular urinary spaces. It was postulated previously and confirmed recently that podocalyxin, a sialomucin, plays a major role in maintaining the urinary space open by virtue of the physicochemical properties of its highly negatively charged ectodomain. This study examined whether the highly conserved cytoplasmic tail of podocalyxin also contributes to the unique organization of podocytes by interacting with the cytoskeletal network found in their cell bodies and foot processes. By immunocytochemistry, it was shown that podocalyxin and the actin binding protein ezrin are co-expressed in podocytes and co-localize along the apical plasma membrane, where they form a co-immunoprecipitable complex. Selective detergent extraction followed by differential centrifugation revealed that some of the podocalyxin cosediments with actin filaments. Moreover, its sedimentation is dependent on polymerized actin and is mediated by complex formation with ezrin. Once formed, podocalyxin/ezrin complexes are very stable, because they are insensitive to actin depolymerization or inactivation of Rho kinase, which is known to be necessary for regulation of ezrin and to mediate Rho-dependent actin organization. These data indicate that in podocytes, podocalyxin is complexed with ezrin, which mediates its link to the actin cytoskeleton. Thus, in addition to its ectodomain, the cytoplasmic tail of podocalyxin also likely contributes to maintaining the unique podocyte morphology.