Translational Studies of the High Molecular Weight Polycyclic Aromatic Hydrocarbon, Dibenzo[ def,p] Chrysene; Carcinogenesis and Metabolism

The objective of this work is to add to the body of translational data between high dose animal model research and the environmentally relevant human metabolism of the persistent pollutant dibenzo[def,p]chrysene (DBC). We furthered the knowledge of gene/exposure interactions by determining the carcinogenesis risk based on Cyp1b1 genotype following in utero DBC exposure in mice, finding no difference in lung carcinogesis by genotype. Cyp1b1 wild-type mice suffered mortality due to aggressive T-Cell Accute Lymphoblastic Leukemia (T-ALL), while Cyp1b1 null and CYP1B1 transgenic mice lack susceptibility to T-ALL. We also determined a link for Cyp1b1 status and fertility status, with a particular vulnerability in Cyp1b1 wild-type male mice fetally exposed to DBC. Female mice were found to have depleted primordial and primary follicle reserves in both Cyp1b1 wild-type and Cyp1b1 null mice. Further research will determine if the extent of depletion is similar across genotypes. In addition, sensitive accelerator mass spectrometry (AMS) was utilized to detect the metabolism of DBC (and BaP) by human volunteers following environmentally relevant exposures. The human PK of DBC validated rodent based PB/PK models generated to represent human metabolism for risk assessment. DBC appeared quickly in plasma, with the majority eliminated quickly over 24 hours and a slower elimination period to 72 hours. The PD of DBC provided evidence that DBC is rapidly biotransformed to metabolites circulating in plasma. The products of three or more bio-transformations (tetrols and beyond) were not present in the plasma, indicating that they are quickly converted to conjugated species and eliminated from the plasma compartment. Urine contained primarily DBC metabolites that had undergone two transformations or more (diols, tetrols, and a putative dione). The majority of these species exist as conjugated metabolites in urine. This body of work furthers the field of PAH translation by addressing disease and disease prevention endpoints from PAH exposure in rodent laboratory research models. Additionally, we are addressing human risk by removing the translation element and directly measuring PAH metabolism in humans from the oral route of exposure by taking a FDA IND "phase 0" approach to pharmacokinetics and pharmacodynamics

Evolutionary and Conservation Genetics of Gopher Frogs, Rana capito and Rana sevosa

Unprecedented growth in the human population has had an indelible impact on the natural landscape. This growth has had serious consequences for many organisms as habitats have been lost and fragmented. Two species of gopher frogs indigenous to the southeastern coastal plain have lost populations throughout much of their natural range. One species, Rana capito, has scattered populations across its fairly wide range and is listed as a species of concern in several states. Another species, Rana sevosa, has been reduced to two small natural populations found only in Mississippi and 4 captive populations in zoos. R. sevosa is listed as federally endangered. Both species require intervention to conserve their numbers. The objectives of this research were 1) to assess the degree of genetic variability and similarity across the range of R. capito and 2) to determine and compare the genetic variability of the two remaining natural populations to the captive populations of R. sevosa. Genetic analyses were performed by genotyping individuals of each population for six microsatellite DNA loci for R. sevosa and one mitochondrial DNA region for R. capito. Genetic variability was analyzed to determine the amount and similarity of genetic variation among populations of R. capito and to determine the interrelatedness of the natural populations of R. sevosa populations as compared to the captive populations. These data will be utilized to understand the current genetic variation of these species with the goal of preserving and enhancing natural population genetic variation

Environmental Pollutants, Mucosal Barriers, and Pathogen Susceptibility; The Case for Aflatoxin B1 as a Risk Factor for HIV Transmission and Pathogenesis

HIV transmission risk is dependent on the infectivity of the HIV+ partner and personal susceptibility risk factors of the HIV− partner. The mucosal barrier, as the internal gatekeeper between environment and self, concentrates and modulates the internalization of ingested pathogens and pollutants. In this review, we summarize the localized effects of HIV and dietary toxin aflatoxin B1 (AFB1), a common pollutant in high HIV burden regions, e.g., at the mucosal barrier, and evidence for pollutant-viral interactions. We compiled literature on HIV and AFB1 geographic occurrences, mechanisms of action, related co-exposures, personal risk factors, and HIV key determinants of health. AFB1 exposure and HIV sexual transmission hotspots geographically co-localize in many low-income countries. AFB1 distributes to sexual mucosal tissues generating inflammation, microbiome changes and a reduction of mucosal barrier integrity, effects that are risk factors for increasing HIV susceptibility. AFB1 exposure has a positive correlation to HIV viral load, a risk factor for increasing the infectivity of the HIV+ partner. The AFB1 exposure and metabolism generates inflammation that recruits HIV susceptible cells and generates chemokine/cytokine activation in tissues exposed to HIV. Although circumstantial, the available evidence makes a compelling case for studies of AFB1 exposure as a risk factor for HIV transmission, and a modifiable new component for combination HIV prevention efforts

Association of plasma aflatoxin with persistent detection of oncogenic human papillomaviruses in cervical samples from Kenyan women enrolled in a longitudinal study

Abstract Background Cervical cancer is caused by oncogenic human papillomaviruses (HR-HPV) and is common among Kenyan women. Identification of factors that increase HR-HPV persistence is critically important. Kenyan women exposed to aflatoxin have an increased risk of HR-HPV detection in cervical specimens. This analysis was performed to examine associations between aflatoxin and HR-HPV persistence. Methods Kenyan women were enrolled in a prospective study. The analytical cohort for this analysis included 67 HIV-uninfected women (mean age 34 years) who completed at least two of three annual study visits and had an available blood sample. Plasma aflatoxin was detected using ultra-high pressure liquid chromatography (UHPLC)-isotope dilution mass spectrometry. Annual cervical swabs were tested for HPV (Roche Linear Array). Ordinal logistic regression models were fitted to examine associations of aflatoxin and HPV persistence. Results Aflatoxin was detected in 59.7% of women and was associated with higher risk of persistent detection of any HPV type (OR = 3.03, 95%CI = 1.08–8.55, P = 0.036), HR-HPV types (OR = 3.63, 95%CI = 1.30-10.13, P = 0.014), and HR-HPV types not included in the 9-valent HPV vaccine (OR = 4.46, 95%CI = 1.13–17.58, P = 0.032). Conclusions Aflatoxin detection was associated with increased risk of HR-HPV persistence in Kenyan women. Further studies, including mechanistic studies are needed to determine if aflatoxin synergistically interacts with HR-HPV to increase cervical cancer risk

MadeenErinEMTHumanInVivo.pdf

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 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 micro-dosing (29 ng, 5 nCi) of [14C]-DBC. This study was made possible with highly sensitive accelerator mass spectrometry (AMS), capable of detecting [14C]-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 apparent elimination rate constant (Kel) of 0.17 ± 0.12 fg*h-1 followed by a slower (β)-phase, with a T1/2 of 41.3 ± 29.8 h and 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 dataset describing pharmacokinetics in humans of a high molecular weight PAH and should be a valuable addition to risk assessment paradigms.To the best of our knowledge, one or more authors of this paper were federal employees when contributing to this work. This is the publisher’s final pdf. The published article is copyrighted by the American Chemical Society and can be found at: https://doi.org/10.1021/tx5003996Keywords: accelerator mass spectrometry, human micro-dosing, polycyclic aromatic hydrocarbon, pharmacokinetics, dibenzo(def\, p)chryseneKeywords: accelerator mass spectrometry, human micro-dosing, polycyclic aromatic hydrocarbon, pharmacokinetics, dibenzo(def\, p)chrysen

MadeenErinEMTHumanInVivo.pdf

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 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 micro-dosing (29 ng, 5 nCi) of [14C]-DBC. This study was made possible with highly sensitive accelerator mass spectrometry (AMS), capable of detecting [14C]-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 apparent elimination rate constant (Kel) of 0.17 ± 0.12 fg*h-1 followed by a slower (β)-phase, with a T1/2 of 41.3 ± 29.8 h and 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 dataset describing pharmacokinetics in humans of a high molecular weight PAH and should be a valuable addition to risk assessment paradigms.To the best of our knowledge, one or more authors of this paper were federal employees when contributing to this work. This is the publisher’s final pdf. The published article is copyrighted by the American Chemical Society and can be found at: https://doi.org/10.1021/tx5003996Keywords: pharmacokinetics, dibenzo(def\, p)chrysene, accelerator mass spectrometry, polycyclic aromatic hydrocarbon, human micro-dosin

Detection and Concentration of Plasma Aflatoxin Is Associated With Detection of Oncogenic Human Papillomavirus in Kenyan Women

Abstract Background Cervical cancer is common in Kenyan women. Cofactors in addition to infection with oncogenic human papillomavirus (HPV) are likely to be important in causing cervical cancer, because only a small percentage of HPV-infected women will develop this malignancy. Kenyan women are exposed to dietary aflatoxin, a potent carcinogen and immunosuppressive agent, which may be such a cofactor. Methods Demographics, behavioral data, plasma, and cervical swabs were collected from 88 human immunodeficiency virus-uninfected Kenyan women without cervical dysplasia. Human papillomavirus detection was compared between women with or without plasma aflatoxin B1-lysine (AFB1-lys) and evaluated in relation to AFB1-lys concentration. Results Valid HPV testing results were available for 86 women (mean age 34.0 years); 49 women (57.0%) had AFB1-lys detected and 37 (43.0%) had none. The AFB1-lys detection was not associated with age, being married, having more than secondary school education, home ownership, living at a walking distance to healthcare ≥60 minutes, number of lifetime sex partners, or age of first sex. The AFB1-lys detection and plasma concentrations were associated with detection of oncogenic HPV types. Conclusions The AFB1-lys positivity and higher plasma AFB1-lys concentrations were associated with higher risk of oncogenic HPV detection in cervical samples from Kenya women. Further studies are needed to determine whether aflatoxin interacts with HPV in a synergistic manner to increase the risk of cervical cancer

Genetic Variation of the Kinases That Phosphorylate Tenofovir and Emtricitabine in Peripheral Blood Mononuclear Cells.

Tenofovir (TFV) disoproxil fumarate and emtricitabine (FTC) are used in combination for HIV treatment and pre-exposure prophylaxis (PrEP). TFV disoproxil fumarate is a prodrug that undergoes diester hydrolysis to TFV. FTC and TFV are nucleoside/nucleotide reverse transcriptase inhibitors that upon phosphorylation to nucleotide triphosphate analogs competitively inhibit HIV reverse transcriptase. We previously demonstrated that adenylate kinase 2, pyruvate kinase, muscle and pyruvate kinase, liver and red blood cell phosphorylate TFV in peripheral blood mononuclear cells (PBMC). To identify the kinases that phosphorylate FTC in PBMC, siRNAs targeted toward kinases that phosphorylate compounds structurally similar to FTC were delivered to PBMC, followed by incubation with FTC and the application of a matrix-assisted laser desorption ionization-mass spectrometry method and ultra high performance liquid chromatography-UV to detect the formation of FTC phosphates. Knockdown of deoxycytidine kinase decreased the formation of FTC-monophosphate, while siRNA targeted toward thymidine kinase 1 decreased the abundance of FTC-diphosphate. Knockdown of either cytidine monophosphate kinase 1 or phosphoglycerate kinase 1 decreased the abundance of FTC-triphosphate. Next-generation sequencing of genomic DNA isolated from 498 HIV-uninfected participants in the HIV Prevention Trials Network 069/AIDS Clinical Trials Group A5305 clinical study, revealed 17 previously unreported genetic variants of TFV or FTC phosphorylating kinases. Of note, four individuals were identified as simultaneous carriers of variants of both TFV and FTC activating kinases. These results identify the specific kinases that activate FTC in PBMC, while also providing further insight into the potential for genetic variation to impact TFV and FTC activation

Genetic Variation of the Kinases That Phosphorylate Tenofovir and Emtricitabine in Peripheral Blood Mononuclear Cells.

Tenofovir (TFV) disoproxil fumarate and emtricitabine (FTC) are used in combination for HIV treatment and pre-exposure prophylaxis (PrEP). TFV disoproxil fumarate is a prodrug that undergoes diester hydrolysis to TFV. FTC and TFV are nucleoside/nucleotide reverse transcriptase inhibitors that upon phosphorylation to nucleotide triphosphate analogs competitively inhibit HIV reverse transcriptase. We previously demonstrated that adenylate kinase 2, pyruvate kinase, muscle and pyruvate kinase, liver and red blood cell phosphorylate TFV in peripheral blood mononuclear cells (PBMC). To identify the kinases that phosphorylate FTC in PBMC, siRNAs targeted toward kinases that phosphorylate compounds structurally similar to FTC were delivered to PBMC, followed by incubation with FTC and the application of a matrix-assisted laser desorption ionization-mass spectrometry method and ultra high performance liquid chromatography-UV to detect the formation of FTC phosphates. Knockdown of deoxycytidine kinase decreased the formation of FTC-monophosphate, while siRNA targeted toward thymidine kinase 1 decreased the abundance of FTC-diphosphate. Knockdown of either cytidine monophosphate kinase 1 or phosphoglycerate kinase 1 decreased the abundance of FTC-triphosphate. Next-generation sequencing of genomic DNA isolated from 498 HIV-uninfected participants in the HIV Prevention Trials Network 069/AIDS Clinical Trials Group A5305 clinical study, revealed 17 previously unreported genetic variants of TFV or FTC phosphorylating kinases. Of note, four individuals were identified as simultaneous carriers of variants of both TFV and FTC activating kinases. These results identify the specific kinases that activate FTC in PBMC, while also providing further insight into the potential for genetic variation to impact TFV and FTC activation

Human Microdosing with Carcinogenic Polycyclic Aromatic Hydrocarbons: <i>In Vivo</i> Pharmacokinetics of Dibenzo[<i>def,p</i>]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[<i>def,p</i>]­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 (<i>C</i>_max = 18.5 ±15.9 fg/mL, <i>T</i>_max= 2.1 ± 1.0 h), whereas the major metabolite was identified as [¹⁴C]-(+/–)-DBC-11,12-diol (<i>C</i>_max= 2.5 ±1.3 fg/mL, <i>T</i>_max= 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 (<i>C</i>_max= 35.8 ± 23.0 pg/pool, <i>T</i>_max = 6–12 h pool). [¹⁴C]-DBC-11,12-diol, of which 97.5% was conjugated, was also identified in urine (<i>C</i>_max = 29.4 ± 11.6 pg/pool, <i>T</i>_max = 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