Polycyclic aromatic hydrocarbons (PAHs) are pollutants created by the incomplete combustion of carbon, and are increasing in the environment largely due to the burning of fossil fuels. PAHs occur as complex mixtures, and some combinations have been shown to cause synergistic developmental toxicity in fish embryos, characterized by pericardial edema and craniofacial malformations. Previous studies have indicated that in the zebrafish model, this toxicity is mediated by the aryl hydrocarbon receptor 2 (AHR2), and enhanced by inhibition of CYP1A activity. In this study, we further examined this interaction of the model PAH and AHR agonist β-naphthoflavone (BNF) with and without the AHR partial agonist/antagonist and CYP1A inhibitor α-naphthoflavone (ANF) to determine 1) whether ANF was acting as an AHR antagonist, 2) what alterations BNF and ANF both alone and in combination had on mRNA expression of the AHR regulated genes cytochrome P450 (cyp) 1a, 1b1, and 1c1, and the AHR repressor (ahrr2) prior to vs. during deformity onset, and 3) compare CYP1A enzyme activity with mRNA induction. Zebrafish embryos were exposed from 24–48 or 24–96 hpf to BNF, 1–100 μg/L, ANF, 1–150 μg/L, a BNF+ANF co-exposure (1 μg/L + 100 μg/L), or a DMSO solvent control. RNA was extracted and examined by quantitative real time PCR. Both BNF and ANF each individually resulted in a dose dependent increase CYP1A, CYP1B1, CYP1C1, and AHRR2 mRNA, confirming their activities as AHR agonists. In the BNF+ANF co-exposures prior to deformity onset, expression of these genes was synergistic, and expression levels of the AHR regulated genes resembled the higher doses of BNF alone. Gene induction during deformities was also significantly increased in the co-exposure, but to a lesser magnitude than prior to deformity onset. EROD measurements of CYP1A activity showed ANF inhibited activity induction by BNF in the co-exposure group; this finding is not predicted by mRNA expression, which is synergistically induced in this treatment. This suggests that inhibition of CYP1A activity may alter metabolism and/or increase the half-life of the AHR agonist(s), allowing for increased AHR activation. This study furthers a mechanistic understanding of interactions underlying PAH synergistic toxicity.
quantitative real-time PCR; CYP1A; CYP1B1; CYP1C1; AHRR; zebrafish; polycyclic aromatic hydrocarbons; aryl hydrocarbon receptor
The aryl hydrocarbon receptor (AhR), a client protein of heat shock protein 90 (HSP90), plays a significant role in polycyclic aromatic hydrocarbon (PAH) induced carcinogenesis. Tobacco smoke, a source of PAHs, activates the AhR leading to enhanced transcription of CYP1A1 and CYP1B1, which encode proteins that convert PAHs to genotoxic metabolites. The main objectives of this study were to determine whether HSP90 inhibitors suppress PAH-mediated induction of CYP1A1 and CYP1B1 or block benzo[a]pyrene (B[a]P) induced formation of DNA adducts. Treatment of cell lines derived from oral leukoplakia (MSK-Leuk1) or esophageal squamous cell carcinoma (KYSE450) with a saline extract of tobacco smoke, B[a]P or dioxin induced CYP1A1 and CYP1B1 transcription resulting in enhanced levels of message and protein. Inhibitors of HSP90 (17-allylamino-demethoxygeldanamycin, 17-AAG; celastrol) suppressed these inductive effects of PAHs. Treatment with 17-AAG and celastrol also caused a rapid and marked decrease in amounts of AhR protein without modulating levels of HSP90. The formation of B[a]P induced DNA adducts in MSK-Leuk1 cells was inhibited by 17-AAG, celastrol and α-naphthoflavone, a known AhR antagonist. The reduction in B[a]P induced DNA adducts was due, at least in part, to reduced metabolic activation of B[a]P. Collectively, these results suggest that 17-AAG and celastrol, inhibitors of HSP90, suppress the activation of AhR-dependent gene expression leading, in turn, to reduced formation of B[a]P induced DNA adducts. Inhibitors of HSP90 may have a role in chemoprevention in addition to cancer therapy.
Heat Shock Protein 90 Inhibitors; Aryl Hydrocarbon Receptor; Tobacco Smoke; Cytochrome P450; DNA adduct
Adaptations to a new environment, such as a polluted one, often involve large modifications of the existing phenotypes. Changes in gene expression and regulation during critical developmental stages may explain these phenotypic changes. Embryos from a population of the teleost fish, Fundulus heteroclitus, inhabiting a clean estuary do not survive when exposed to sediment extract from a site highly contaminated with polycyclic aromatic hydrocarbons (PAHs) while embryos derived from a population inhabiting a PAH polluted estuary are remarkably resistant to the polluted sediment extract. We exposed embryos from these two populations to surrogate model PAHs and analyzed changes in gene expression, morphology, and cardiac physiology in order to better understand sensitivity and adaptive resistance mechanisms mediating PAH exposure during development.
The synergistic effects of two model PAHs, an aryl hydrocarbon receptor (AHR) agonist (β-naphthoflavone) and a cytochrome P4501A (CYP1A) inhibitor (α-naphthoflavone), caused significant developmental delays, impaired cardiac function, severe morphological alterations and failure to hatch, leading to the deaths of reference embryos; resistant embryos were mostly unaffected. Unexpectedly, patterns of gene expression among normal and moderately deformed embryos were similar, and only severely deformed embryos showed a contrasting pattern of gene expression. Given the drastic morphological differences between reference and resistant embryos, a surprisingly low percentage of genes, 2.24% of 6,754 analyzed, show statistically significant differences in transcript levels during late organogenesis between the two embryo populations.
Our study demonstrates important contrasts in responses between reference and resistant natural embryo populations to synergistic effects of surrogate model PAHs that may be important in adaptive mechanisms mediating PAH effects during fish embryo development. These results suggest that statistically significant changes in gene expression of relatively few genes contribute to the phenotypic changes and large morphological differences exhibited by reference and resistant populations upon exposure to PAH pollutants. By correlating cardiac physiology and morphology with changes in gene expression patterns of reference and resistant embryos, we provide additional evidence for acquired resistance among embryos whose parents live at heavily contaminated sites.
Development; Genomics; Embryos; Adaptation; Evolution; Fish; Transcriptomics
To evaluate the association of polycyclic aromatic hydrocarbon (PAH) exposure in esophageal epithelial tissue and esophageal squamous cell carcinoma (ESCC) case status in an ESCC case-control study in a high-risk population in northeastern Iran.
Immunohistochemical staining of tissue microarrays (TMAs) of non-tumoral esophageal biopsies from ESCC cases and control subjects. Immunohistochemistry was performed using monoclonal antibodies 8E11 and 5D11, raised against benzo[a]pyrene (B[a]P) diol epoxide (BPDE)-I-modified guanosine and BPDE-I-modified DNA, respectively. Staining intensity was quantified by image analysis, and the average staining in three replicates was calculated.
Rural region in northeastern Iran.
Cases were patients with biopsy-proven ESCC. Controls were GI clinic patients with no endoscopic or biopsy evidence of ESCC.
Main outcome measure
Adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) for the association between antibody staining intensity and ESCC case status.
Cultured ESCC cells exposed to B[a]P in vitro showed dose-dependent staining with 8E11, but not with 5D11. With 8E11, sufficient epithelial tissue was available in the TMA cores to analyze 91 cases and 103 controls. Compared to the lowest quintile of 8E11 staining in the controls, adjusted ORs (95% CIs) for the 2nd to 5th quintiles were 2.42, 5.77, 11.3, and 26.6 (5.21–135), respectively (P for trend < 0.001). With 5D11, 89 cases and 101 controls were analyzed. No association between staining and case status was observed (ORs (95% CIs) for the 2nd to 5th quintiles were 1.26, 0.88, 1.06, and 1.63 (0.63–4.21), P for trend = 0.40).
Dramatically higher levels of 8E11 staining were observed in non-tumoral esophageal epithelium from ESCC patients than from control subjects. This finding strengthens the evidence for a causal role for PAHs in esophageal carcinogenesis in northeastern Iran.
esophageal squamous cell carcinoma; polycyclic aromatic hydrocarbons; immunohistochemistry; tissue microarray
The aryl hydrocarbon receptor (AhR), a ligand-activated member of the basic-helix-loop-helix family of transcription factors, plays a significant role in polycyclic aromatic hydrocarbon (PAH) induced carcinogenesis. In the upper aerodigestive tract of humans, tobacco smoke, a source of PAHs, activates the AhR leading to increased expression of CYP1A1 and CYP1B1, which encode proteins that convert PAHs to genotoxic metabolites. Inhibitors of Hsp90 ATPase cause a rapid decrease in levels of AhR, an Hsp90 client protein, and thereby block PAH-mediated induction of CYP1A1 and CYP1B1. The main objective of this study was to determine whether Zyflamend, a polyherbal preparation, suppressed PAH-mediated induction of CYP1A1 and CYP1B1 and inhibited DNA adduct formation and mutagenesis. We also investigated whether carnosol, one of multiple phenolic antioxidants in Zyflamend, had similar inhibitory effects. Treatment of cell lines derived from oral leukoplakia (MSK-Leuk1) and skin (HaCaT) with benzo[a]pyrene (B[a]P), a prototypic PAH, induced CYP1A1 and CYP1B1 transcription, resulting in enhanced levels of message and protein. Both Zyflamend and carnosol suppressed these effects of B[a]P. Notably, both Zyflamend and carnosol inhibited Hsp90 ATPase activity and caused a rapid reduction in AhR levels. The formation of B[a]P induced DNA adducts and mutagenesis were also inhibited by Zyflamend and carnosol. Collectively, these results show that Zyflamend and carnosol inhibit Hsp90 ATPase leading to reduced levels of AhR, suppression of B[a]P-mediated induction of CYP1A1 and CYP1B1 and inhibition of mutagenesis. Carnosol-mediated inhibition of Hsp90 ATPase activity can help explain the chemopreventive activity of herbs such as Rosemary, which contain this phenolic antioxidant.
The aryl hydrocarbon receptor (AhR) is part of a powerful signaling system that is triggered by xenobiotic agents such as polychlorinated hydrocarbons (PCHs) and polycyclic aromatic hydrocarbons (PAHs). Although activation of the AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or certain polycyclic aromatic hydrocarbons (PAHs) can lead to immunosuppression, there is also increasing evidence that the AhR regulates certain normal developmental processes. In this study, we asked whether the AhR plays a role in host resistance using murine listeriosis as an experimental system. Our data clearly demonstrate that AhR null C57BL/6J mice (AhR−/−) are more susceptible to listeriosis than AhR heterozygous (AhR+/−) littermates when inoculated i.v. with log-phase L. monocytogenes. AhR−/− mice exhibited greater numbers of CFU of L. monocytogenes in the spleen and liver, and greater histopathological changes in the liver than AhR+/− mice. Serum levels of IL-6, MCP-1, IFN-γ, and TNF-α were comparable between L. monocytogenes-infected AhR−/− and AhR+/− mice. Increased levels of IL-12 and IL-10 were observed in L. monocytogenes-infected AhR−/− mice. No significant difference was found between AhR+/− and AhR−/− macrophages ex vivo with regard to their ability to ingest and inhibit intracellular growth of L. monocytogenes. Intracellular cytokine staining of CD4+ and CD8+ splenocytes for IFN-γ and TNF-α revealed comparable T-cell mediated responses in AhR−/− and AhR+/− mice. Previously infected AhR−/− and AhR+/− mice both exhibited enhanced resistance to reinfection with L. monocytogenes. These data provide the first evidence that AhR is required for optimal resistance, but is not essential for adaptive immune response to L. monocytogenes infection.
aryl hydrocarbon receptor (AhR); L. monocytogenes; innate immunity; adaptive immunity
Widespread contamination of aquatic systems with polycyclic aromatic hydrocarbons (PAHs) has led to concern about effects of PAHs on aquatic life. Some PAHs have been shown to cause deformities in early life stages of fish that resemble those elicited by planar halogenated aromatic hydrocarbons (pHAHs) that are agonists for the aryl hydrocarbon receptor (AHR). Previous studies have suggested that activity of cytochrome P4501A, a member of the AHR gene battery, is important to the toxicity of pHAHs, and inhibition of CYP1A can reduce the early-life-stage toxicity of pHAHs. In light of the effects of CYP1A inhibition on pHAH-derived toxicity, we explored the impact of both model and environmentally relevant CYP1A inhibitors on PAH-derived embryotoxicity. We exposed Fundulus heteroclitus embryos to two PAH-type AHR agonists, β-naphthoflavone and benzo(a)pyrene, and one pHAH-type AHR agonist, 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126), alone and in combination with several CYP1A inhibitors. In agreement with previous studies, coexposure of embryos to PCB-126 with the AHR antagonist and CYP1A inhibitor α-naphthoflavone decreased frequency and severity of deformities compared with embryos exposed to PCB-126 alone. In contrast, embryos coexposed to the PAHs with each of the CYP1A inhibitors tested were deformed with increased severity and frequency compared with embryos dosed with PAH alone. The mechanism by which inhibition of CYP1A increased embryotoxicity of the PAHs tested is not understood, but these results may be helpful in elucidating mechanisms by which PAHs are embryotoxic. Additionally, these results call into question additive models of PAH embryotoxicity for environmental PAH mixtures that contain both AHR agonists and CYP1A inhibitors.
α-naphthoflavone; aryl hydrocarbon receptor; benzo(a)pyrene; β-naphthoflavone; cytochrome P4501A; deformity; fluoranthene; Fundulus heteroclitus; polychlorinated biphenyls; polycyclic aromatic hydrocarbons
Drinking maté, common in southern South America, may increase the risk of esophageal squamous cell carcinoma (ESCC). In 2006, we found high but variable polycyclic aromatic hydrocarbon (PAH) content in commercial yerba maté samples from eight Brazilian brands. The PAH content of new samples from the same brands, purchased in 2008, and four brands from a single manufacturer processed in different ways, obtained in 2010, were quantified to determine whether PAH concentration was still high, PAH content variation was brand specific, and whether processing method affects PAH content of commercial yerba maté. Concentrations of individual PAHs were quantified using gas chromatography/mass spectrometry with deuterated PAHs as internal standards. Median total PAH concentration was 1500 ng/g (range: 625 to 3710 ng/g) and 1090 ng/g (621 to 1990 ng/g) in 2008 and 2010 samples, respectively. Comparing 2006 and 2008 samples, some brands had high PAH concentrations in both years, while PAH concentration changed considerably in others. Benzo[a]pyrene concentrations ranged from 11.9 to 99.3 ng/g and 5.11 to 21.0 ng/g in 2008 and 2010 samples, respectively. The 2010 sample processed without touching smoke had the lowest benzo[a]pyrene content. These results support previous findings of very high total and carcinogenic PAH concentrations in yerba maté, perhaps contributing to the high incidence of ESCC in southern South America. The large PAH content variation by brand, batch and processing method suggests it may be possible to reduce the content of carcinogenic PAHs in commercial yerba maté, making it a healthier beverage.
Polycyclic Aromatic Hydrocarbons; Yerba Maté; Carcinogens; Esophageal Cancer; Benzo(α)pyrene; Processing Method; Lifestyle
Exposure of developing fish to polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs) results in a suite of defects including cardiac malformation, pericardial and yolk sac edema, craniofacial defects, and hemorrhaging. Several populations of Atlantic killifish or mummichog (Fundulus heteroclitus) on the Atlantic coast of the United States are resistant to the developmental and acute toxicity caused by PAHs and HAHs; this has made Fundulus a valuable model for studying aryl hydrocarbon sensitivity and adaptation. In order to further increase the utility of Fundulus, better understanding of the components of the molecular pathways governing aryl hydrocarbon response in Fundulus is required. The aryl hydrocarbon receptor (AHR) is known to mediate many of the toxic responses to PAHs and HAHs. A single AHR has been identified in mammals, but Fundulus has two AHRs and their relative roles are not clear. In the current study, translation-blocking and splice-junction morpholino gene knockdown was used to determine the roles of AHR1 and AHR2 in mediating cardiac teratogenesis induced by β-naphthoflavone (BNF), benzo[k]fluoranthene (BkF), and 3, 3′, 4, 4′, 5-pentachlorobiphenyl (PCB-126). Here we report that AHR2 and not AHR1 knockdown resulted in rescue of teratogenicity induced by BNF, BkF, and PCB-126. These data demonstrate that AHR2 is the primary mediator of cardiac teratogenesis caused by multiple aryl hydrocarbons in Fundulus and suggest that suppression of the AHR pathway through modulation of AHR2 is a plausible mechanism for PAH resistance in adapted fish. Additionally, this is the first reported use of splice-junction morpholinos in Fundulus.
aryl hydrocarbon receptor; polycyclic aromatic hydrocarbon; PCB-126; morpholino; Fundulus heteroclitus; adaptation
The aryl hydrocarbon (dioxin) receptor (AhR) is a ligand-dependent transcription factor that produces a wide range of biological and toxic effects in many species and tissues. Whereas the best-characterized high-affinity ligands include structurally related halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs), the AhR is promiscuous and can also be activated by structurally diverse exogenous and endogenous chemicals. However, little is known about how these diverse ligands actually bind to and activate the AhR. Utilizing AhR ligand binding, DNA binding, and reporter gene expression assays, we have identified a novel ligand-selective antagonist (CH223191) that preferentially inhibits the ability of some classes of AhR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin and related HAHs), but not others (PAHs, flavonoids, or indirubin), to bind to and/or activate the AhR and AhR signal transduction. HAH-specific antagonism of AhR-dependent reporter gene expression by CH223191 was observed with mouse, rat, human, and guinea pig cell lines. Ligand- and species-selective antagonism was also observed with the AhR antagonists 3′-methoxy-4′-nitroflavone and 6,2′,4′,-trimethoxyflavone. Our results suggest that the differences in the binding by various ligands to the AhR contribute to the observed structural diversity of AhR ligands and could contribute in ligand-specific variation in AhR functionality and the toxic and biological effects of various classes of AhR agonists.
2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; Ah receptor; beta-naphthoflavone; CH223191
Maternal smoking during pregnancy is associated with a variety of adverse neonatal outcomes including altered reproductive performance. Herein we provide molecular evidence for a pathway involved in the elimination of the female germline due to prepregnancy and/or lactational exposure to polycyclic aromatic hydrocarbons (PAHs), environmental toxicants found in cigarette smoke. We show that ovaries of offspring born to mice exposed to PAHs contained only a third of the ovarian follicle pool compared with offspring of unexposed female mice. Activation of the cell death pathway in immature follicles of exposed females was mediated by the aryl hydrocarbon receptor (Ahr), as ovarian reserve was fully rescued by maternal cotreatment with the Ahr antagonist, resveratrol, or by inactivation of the Ahr gene. Furthermore, in response to PAHs, Ahr-mediated activation of the harakiri, BCL2 interacting protein (contains only BH3 domain), was necessary for execution of cell death. This pathway appeared to be conserved between mouse and human, as xenotransplanted human ovarian cortex exposed to PAHs responded by activation of the identical cell death cascade. Our data indicate that maternal exposure to PAHs prior to pregnancy and/or during lactation compromises ovarian reserve of female offspring, raising the concern about the transgenerational impact of maternal smoking on ovarian function in the human.
Fundulus heteroclitus (Atlantic killifish) found at the Atlantic Wood Industries Superfund site on the Elizabeth River (ER) in Portsmouth, VA (USA), have been shown to be resistant to the teratogenic effects of creosote-contaminated sediments found at this highly contaminated site. Many of the polycyclic aromatic hydrocarbons (PAHs) found at the ER are known to activate the aryl hydrocarbon receptor (AHR), and are thought to mediate their toxic effects through this pathway. Activation of the AHR results in the induction of several Phase I and II metabolic enzymes. It has been previously shown that the AHR of killifish from the ER are refractory to induction by AHR agonists. To more fully characterize this altered AHR response, we exposed embryos from the ER and from a reference site on King's Creek, VA (KC) to two PAHs, benzo[α]pyrene (BaP) and benzo[k]fluoranthene (BkF), and to the dioxin-like compound (DLC), 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). We compared their developmental and molecular responses by screening the embryos for CYP1A enzyme activity, cardiac deformities, and mRNA expression of CYP1A, CYP1B1, CYP1C1, and AHR2. Basal gene expression of both CYP1A and CYP1B1 was 40% higher in the KC control embryos compared to those from the ER, while AHR2 and CYP1C1 were not significantly different between the populations. Exposure of KC embryos to BaP, BkF, and PCB126 induced CYP1A activity and cardiac deformities. In contrast, CYP1A activity was induced in ER embryos only in response to BkF exposure, although this induction in ER embryos was significantly lower than that observed in KC fish at comparable concentrations. ER embryos did not develop cardiac deformities in response to any of the chemicals tested. CYP1A, CYP1B1 and CYP1C1 mRNA were all significantly induced in the KC embryos after exposure to BaP, BkF and PCB126. Exposure to BaP and BkF in ER embryos resulted in a significant induction of CYP1A mRNA, albeit significantly lower than observed in KC fish. Interestingly, BaP exposure resulted in induction of CYP1B1 at comparable levels in embryos from both populations. CYP1s were not induced in ER embryos in response to PCB126, nor was CYP1C1 for any treatment examined. Additionally, AHR2 was not significantly induced for any of the treatment groups. This study further characterizes the AHR response in killifish, and provides greater insight into the adapted ER phenotype. The ER adaptation involves the suppression of normal AHR-inducible gene expression for all three CYP1 genes, and therefore is likely an alteration in AHR signaling or control.
Fundulus heteroclitus; Elizabeth River; cytochrome P450-1; polycyclic aromatic hydrocarbon; Benzo[a]pyrene; benzo[k]fluoranthene; 3,3′,4,4′,5-pentachlorobiphenyl
Exposure to traffic-related air pollutants, including polycyclic aromatic hydrocarbons (PAHs), can induce asthma. However, the effects of early repeated PAH exposure over time on different asthma phenotypes have not been examined.
To assess associations between repeated PAH exposure, measured from prenatal personal and residential indoor monitors in children's homes, and asthma in an inner-city cohort.
Prenatal exposure was assessed by personal air monitoring during 48 hours and exposure at 5 to 6 years of age by 2-week residential monitoring in the Columbia Center for Children's Environmental Health cohort. PAH was dichotomized into pyrene (representative semivolatile PAH) and the sum of 8 nonvolatile PAHs. High exposure to each was defined as measures above the median at both repeated time points. Asthma and wheeze were determined by validated questionnaires at ages 5 to 6 years. Children with specific IgE levels greater than 0.35 IU/mL to any of 5 indoor allergens were considered seroatopic.
Among all 354 children, repeated high exposure to pyrene was associated with asthma (odds ratio [OR], 1.90; 95% confidence interval [CI], 1.13-3.20). Among 242 nonatopic children, but not those sensitized to indoor allergens (n = 87) or with elevated total IgE levels (n = 171), high pyrene levels were associated positively with asthma (OR, 2.89; 95% CI, 1.77-5.69), asthma medication use (OR, 2.28; 95% CI, 1.13-4.59), and emergency department visits for asthma (OR, 2.43; 95% CI, 1.20-4.91). Associations between the levels of the 8 nonvolatile PAHs and asthma were not observed, even when stratifying by seroatopy.
Nonatopic children may be more susceptible to the respiratory consequences of early pyrene exposures.
Populations of organisms that are chronically exposed to high levels of chemical contaminants may not suffer the same sublethal or lethal effects as naive populations, a phenomenon called resistance. Atlantic tomcod (Microgadus tomcod) from the Hudson River, New York, are exposed to high concentrations of polycyclic aromatic hydrocarbons (PAHs) and bioaccumulate polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs). They have developed resistance to PCBs and PCDDs but not to PAHs. Resistance is largely heritable and manifests at early-life-stage toxic end points and in inducibility of cytochrome P4501A (CYP1A) mRNA expression. Because CYP1A induction is activated by the aryl hydrocarbon receptor (AHR) pathway, as are most toxic responses to these compounds, we sought to determine the geographic extent of resistance to CYP1A mRNA induction by PCBs in the Hudson River tomcod population. Samples of young-of-the-year tomcod were collected from seven locales in the Hudson River, extending from the Battery at river mile 1 (RM 1) to RM 90, and from the Miramichi River, New Brunswick, Canada. Laboratory-reared offspring of tomcod adults from Newark Bay, in the western portion of the Hudson River estuary, were also used in this study. Fish were partially depurated in clean water and intraperitoneally injected with 10 ppm coplanar PCB-77, 10 ppm benzo[a]pyrene (BaP), or corn oil vehicle, and levels of CYP1A mRNA were determined. CYP1A was significantly inducible by treatment with BaP in tomcod from the Miramichi River, from laboratory-spawned offspring of Newark Bay origin, and from all Hudson River sites spanning 90 miles of river. In contrast, only tomcod from the Miramichi River displayed significantly induced CYP1A mRNA expression when treated with PCB-77. Our results suggest that the population of tomcod from throughout the Hudson River estuary has developed resistance to CYP1A inducibility and probably other toxicities mediated by the AHR pathway. Tomcod from the Hudson River may represent the most geographically expansive population of vertebrates with resistance to chemical pollutants that has been characterized.
AHR; Atlantic tomcod; CYP1A; evolutionary change; genetic adaptation; Hudson River; PCBs; resistance
Polycyclic aromatic hydrocarbons (PAHs), derived largely from fossil fuels and their combustion, are pervasive contaminants in rivers, lakes, and nearshore marine habitats. Studies after the Exxon Valdez oil spill demonstrated that fish embryos exposed to low levels of PAHs in weathered crude oil develop a syndrome of edema and craniofacial and body axis defects. Although mechanisms leading to these defects are poorly understood, it is widely held that PAH toxicity is linked to aryl hydrocarbon receptor (AhR) binding and cytochrome P450 1A (CYP1A) induction. Using zebrafish embryos, we show that the weathered crude oil syndrome is distinct from the well-characterized AhR-dependent effects of dioxin toxicity. Blockade of AhR pathway components with antisense morpholino oligonucleotides demonstrated that the key developmental defects induced by weathered crude oil exposure are mediated by low-molecular-weight tricyclic PAHs through AhR-independent disruption of cardiovascular function and morphogenesis. These findings have multiple implications for the assessment of PAH impacts on coastal habitats.
cardiovascular function; fish development; non-point source pollution; oil spill
Atlantic killifish (Fundulus heteroclitus) from the Atlantic Wood Superfund site on the Elizabeth River (ER), VA are dramatically resistant to the acute toxicity and teratogenesis caused by polycyclic aromatic hydrocarbons (PAHs). To understand the consequences of adaptation to chronic PAH pollution, we have attempted to further define the chemical tolerance associated with this resistance. An important component of the PAH adaptation of ER fish is the dramatic down-regulation of the aryl hydrocarbon receptor (AHR) pathway, resulting in decreased cytochrome p450 (CYP) 1 activity. Herein, we compared the susceptibility to several insecticides of ER fish to that of reference site fish; use of these chemicals as probes of the resistance will help to demonstrate if the contaminant adaptation exhibited by ER fish is broad or narrow and AHR-focused. We hypothesized that ER fish would be less susceptible to the organophosphate chlorpyrifos (activated by CYP) and more susceptible to the pyrethroid permethrin (detoxified by CYP). Comparison of acute toxicity in 5-day-old larvae supported this hypothesis for chlorpyrifos. As expected in KC, chemical up-regulation of CYP by co-exposure to β-naphthoflavone (BNF) enhanced the susceptibility of KC but it did not affect ER larvae. Unexpectedly, ER larvae were much less susceptible to permethrin than KC larvae. However, co-exposure to BNF greatly decreased the susceptibility of KC larvae, indicating that metabolism of permethrin by CYP was protective. Additionally, fish from each population were compared for susceptibility to the carbamate carbaryl, an acute neurotoxicant and weak AHR agonist that induces teratogenesis similar to that caused by PAHs. ER embryos and larvae were less susceptible than KC fish. These results suggest that the adaptive phenotype of ER fish is multi-faceted and that aspects other than CYP response are likely to greatly affect their response to contaminants.
Fundulus heteroclitus; adaptation; polycyclic aromatic hydrocarbon; pesticides; Elizabeth River
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants often present in aquatic systems as complex mixtures. Embryonic fish are sensitive to the developmental toxicity of some PAHs, but the exact mechanisms involved in this toxicity are still unknown. This study explored the role of the aryl hydrocarbon receptor (AHR) in the oxidative stress response of zebrafish to the embryotoxicity of select PAHs. Embryos were exposed to two PAHs, benzo[k]fluoranthene (BkF; a strong AHR agonist) and fluoranthene (FL; a cytochrome P4501A (CYP1A) inhibitor), alone and in combination. CYP1A, CYP1B1, CYP1C1, and redox-responsive genes glutathione s-transferase pi 2 (GSTp2), glutathione peroxidase 1 (GPx1), the glutamate-cysteine ligase catalytic subunit (GCLc), MnSOD and CuZnSOD mRNA expression was examined. CYP1 activity was measured via an in vivo ethoxyresorufin-O-deethlyase (EROD) activity assay, and the area of the pericardium was measured as an index of cardiotoxicity. BkF or FL alone caused no deformities whereas BkF + FL resulted in extreme pericardial effusion. BkF induced CYP activity above controls and co-exposure with FL inhibited this activity. BkF induced expression of all three CYPs, GSTp2, and GCLc. BkF + FL caused greater than additive induction of the three CYPs, GSTp2, GPx1, and GCLc but had no effect on MnSOD or CuZnSOD. AHR2 knockdown protected against the cardiac deformities caused by BkF + FL and significantly inhibited the induction of the CYPs, GSTp2, GPx1 and GCLc after BkF + FL compared to non-injected controls. These results further show the protective role of AHR2 knockdown against cardiotoxic PAHs and the role of AHR2 as a mediator of redox-responsive gene induction.
zebrafish; polycyclic aromatic hydrocarbons (PAHs); oxidative stress; morpholino; aryl hydrocarbon receptor (AHR)
The high lung cancer rate in Xuan Wei, China, is associated with smoky coal use in unvented homes, but not with wood or smokeless coal use. Smoky coal combustion emits higher polycyclic aromatic hydrocarbon (PAH) concentrations than wood combustion. This study used DNA adducts as biomarkers for human exposure to PAH from combustion emissions. DNA adducts were determined by enzyme-linked immunosorbent assays (ELISA) in placentas and peripheral and cord white blood cells (WBC) from Xuan Wei women burning smoky coal or wood and from Beijing women using natural gas. Color ELISA gave positive results in 58, 47, and 5% of the placentas from Xuan Wei women burning smoky coal without and with chimneys, and from Beijing women, respectively. Fluorescence ELISA indicated that 46, 65, 56, and 25% of placentas were positive from Xuan Wei women who lived in houses without and with chimneys, Xuan Wei women burning wood, and Beijing controls, respectively. Peripheral WBC samples were positive in 7/9, 8/9, and 3/9 for the Xuan Wei women who lived in houses without and with chimneys and Beijing women, respectively. PAH-DNA adducts were detected in a higher percentage of placentas from Xuan Wei women living in houses exposed to smoky coal or wood emissions than from those of the Beijing controls. No dose-response relationship was observed between the air benzo[alpha]pyrene concentrations and DNA adduct levels or percentage of detectable samples. The results suggest that DNA adducts can be used as a qualitative biomarker to assess human exposure to combustion emissions.
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxic effects of halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), and other structurally diverse ligands. While HAHs are several orders of magnitude more potent in producing AhR-dependent biochemical effects than PAHs or other AhR agonists, only the HAHs have been observed to produce AhR-dependent toxicity in vivo. Here we have characterized the dissociation of a prototypical HAH ligand ([3H] 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) and PAH-like ligand ([3H] β-naphthoflavone [βNF]) from the guinea pig, hamster, mouse, and rat hepatic cytosolic AhR in order to elucidate the relationship between the apparent ligand-binding affinities and the divergent potency of these chemicals. Both compounds dissociated very slowly from the AhR with the amount of specific binding remaining at 96 h ranging from 53% to 70% for [3H]TCDD and 26% to 85% for [3H] βNF, depending upon the species examined. The rate of ligand dissociation was unaffected by protein concentration or incubation temperature. Preincubation of cytosol with 2,3,7,8-tetrachlorodibenzofuran, carbaryl, or primaquine, prior to the addition of [3H]TCDD, shifted the apparent IC50 of these compounds as competitive AhR ligands by ∼10- to 50-fold. Our results support the need for reassessment of previous AhR ligand-binding affinity calculations and competitive binding analysis since these measurements are not carried out at equilibrium binding conditions. Our studies suggest that AhR binding affinity/occupancy has little effect on the observed differences in the persistence of gene expression by HAHs and PAHs.
Ah receptor; 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; β-naphthoflavone
Occupational and environmental exposure to polycyclic aromatic hydrocarbons (PAHs) has been suggested to provoke inflammatory and/or allergic disorders, including asthma, rhinitis, and dermatitis. The molecular mechanisms of this PAH-mediated inflammation remain to be clarified. Previous studies implied the involvement of PAHs as irritants and allergens, with the reactive oxygen species generated from the oxygenated PAHs believed to be an exacerbating factor. It is also possible that PAHs contribute to the pathogenesis through activation of aryl-hydrocarbon receptor (AhR)-mediated transcription, since PAHs are potent inducers of the AhR. To address this point, we generated transgenic mouse lines expressing the constitutive active form of the AhR in keratinocytes. In these lines of mice, the AhR activity was constitutively enhanced in the absence of ligands, so that any other direct effects of PAHs and their metabolites could be ignored. At birth, these transgenic mice were normal, but severe skin lesions with itching developed postnatally. The skin lesions were accompanied by inflammation and immunological imbalance and resembled typical atopic dermatitis. We demonstrate that constitutive activation of the AhR pathway causes inflammatory skin lesions and suggests a new mechanism for the exacerbation of inflammatory diseases after exposure to occupational and environmental xenobiotics.
The aryl hydrocarbon receptor (AhR, also called the dioxin receptor) plays crucial roles in toxicologic responses of animals to environmental pollutants, especially to halogenated and polycyclic aromatic hydrocarbons. To achieve direct, continuous risk assessment of air pollution using biological systems, we generated transgenic sensor mice that produce secreted alkaline phosphatase (SEAP) under the control of AhR.
To characterize responses of the mice to AhR agonists, sensor mice were orally administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3MC), benzo[a]pyrene (B[a]P), or β-naphthoflavone (BNF), and serum levels of SEAP were evaluated. To monitor air pollution caused by cigarette smoke, we placed the mice each day in an experimental smoking room, and evaluated activity of serum SEAP for up to 4 days. Activation of AhR in individual organs was also examined by reverse transcription–polymerase chain reaction (RT-PCR) analysis of SEAP.
In response to oral exposure to TCDD, sensor mice exhibited dramatic and sustained activation of AhR. The mice also responded sensitively to 3MC, B[a]P, and BNF. Activation of AhR was dose dependent, and the liver was identified as the main responding organ. After exposure to the smoking environment, sensor mice consistently exhibited transient, reversible activation of AhR. RT-PCR analysis of SEAP revealed that activation of AhR occurred predominantly in the lung.
We are the first laboratory to demonstrate successfully direct, comprehensive monitoring of air pollution using genetically engineered mammals. The established system would be useful for real risk assessment of halogenated and polycyclic aromatic hydrocarbons in the air, especially in smoking environments.
aromatic hydrocarbon; aryl hydrocarbon receptor (AhR); cigarette smoke; dioxin-responsive element (DRE); secreted alkaline phosphatase (SEAP); transgenic mouse
Industrialized regions in Poland are characterized by high ambient pollution, including polycyclic aromatic hydrocarbons (PAHs) from coal burning for industry and home heating. In experimental bioassays, certain PAHs are transplacental carcinogens and developmental toxicants. Biologic markers can facilitate evaluation of effects of environmental PAHs on the developing infant. We measured the amount of PAHs bound to DNA (PAH-DNA adducts) in maternal and umbilical white blood cells. The cohort consisted of 70 mothers and newborns from Krakow, Poland, an industrialized city with elevated air pollution. Modulation of adduct levels by genotypes previously linked to risk of lung cancer, specifically glutathione S-transferase MI (GSTM1) and cytochrome P4501A1 (CYP1A1) Msp restriction fragment length polymorphism (RFLP), was also investigated. There was a dose-related increase in maternal and newborn adduct levels with ambient pollution at the women's place of residence among subjects who were not employed away from home (p < or = 0.05). Maternal smoking (active and passive) significantly increased maternal (p < or = 0.01) but not newborn adduct levels. Neither CYP1A1 Msp nor GSTM1 polymorphisms was associated with maternal adducts. However, adducts were significantly higher in newborns heterozygous or homozygous for the CYP1A1 Msp RFLP compared to newborns without the RFLP (p = 0.04). Results indicate that PAH-induced DNA damage in mothers and newborns is increased by ambient air pollution. In the fetus, this damage appears to be enhanced by the CYP1A1 Mspl polymorphism.
The leading cause of lung cancer is exposure to cigarette smoke and other environmental pollutants, which include formaldehyde, acrolein, benzene, dioxin, and polycyclic aromatic hydrocarbons (PAHs). PAHs and dioxins are exogenous ligands that directly bind to the aryl hydrocarbon receptor (AhR), a transcription factor that activates xenobiotic metabolism, histone modification (an important step in DNA methylation), and, ultimately, tumorigenesis. Here we summarize the current understanding of AhR and its role in the development of lung cancer, including its influence on cell proliferation, angiogenesis, inflammation, and apoptosis.
Aryl hydrocarbon receptor; lung cancer; tumorigenesis; dioxin; polycyclic aromatic hydrocarbons; cigarette smoke
There has been relatively little bioanalytical effect based monitoring conducted using samples derived from polyurethane foam (PUF) passive air samplers. Combining these techniques may provide a more convenient and cost effective means of monitoring the potential for biological effects resulting from exposure to complex mixtures in a range of scenarios. Seasonal polycyclic aromatic hydrocarbon (PAH) levels were monitored at sites around Australia using direct chemical analysis. In addition, both indirect acting genotoxicity (umuC assay) and aryl hydrocarbon receptor (AhR) activity (chemically activated fluorescent gene expression [CAFLUX assay]), which are effects potentially relevant to subsequent carcinogenesis for these compounds, were measured. The levels of PAHs as well as genotoxicity and AhR activity were all higher in winter compared to summer and for sites in urban capital cities compared to other locations. Statistically significant relationships were found between the levels of PAHs and both genotoxicity and AhR activity. The dominant contributors to the total AhR activity, were found to be for compounds which are not resistant to H2SO4/silica gel treatment and were relatively rapidly metabolised that is consistent with a PAH type response. Relative potency estimates for individual PAHs determined for the first time on the CAFLUX assay were used to estimate the proportion of total AhR activity (≤ 3.0%) accounted by PAHs monitored. Observed responses are thus largely due to non-quantified AhR active compounds.
Passive air sampling; Polyurethane foam (PUF); Polycyclic aromatic hydrocarbons; Aryl hydrocarbon receptor activity; Genotoxicity
The aim of the Teplice Program is to investigate and assess the impact of air pollution on the health of the population in the district of Teplice, Czech Republic. Characterization of the air pollutants demonstrated unusually high concentrations during winter inversions of fine particles dominated by acidic sulfates, genotoxic organic compounds, and toxic trace elements. The major source of airborne fine particles is the burning of coal for heating and power. Human exposure and biomarker studies demonstrated large seasonal variations in air pollution within the Teplice District and higher seasonal average pollution levels than the comparative district, Prachatice. Personal exposures to fine particles and organic carcinogens [e.g., polycyclic aromatic hydrocarbons (PAH)] were correlated with excretion of PAH metabolites in urine, several trace metals in blood, and DNA adducts in white blood cells. Respiratory and neurobehavioral studies of school children were conducted using questionnaires and clinical measures. A significantly higher prevalence of adverse respiratory symptoms and decreased lung function were found in the Teplice district than in Prachatice. The neurobehavioral studies indicated significantly higher teacher referrals for clinical assessment in Teplice, but the majority of objective performance measures did not differ. Reproductive studies were conducted in both males and females. A study of the effects of exposure on pregnancy and birth found an excess prevalence of low birth weight and premature births in Teplice; these adverse effects were more common in infants conceived in the winter and whose mothers were smokers. Based on questionnaires and medical examination, the reproductive development of young men was not different between districts and seasons, however, measures of semen quality suggest that exposure to high levels of air pollution are associated with transient decrements in semen quality.