Developmental Deltamethrin Exposure Causes Persistent Changes in Dopaminergic Gene Expression, Neurochemistry, and Locomotor Activity in Zebrafish.

Pyrethroids are commonly used insecticides that are considered to pose little risk to human health. However, there is an increasing concern that children are more susceptible to the adverse effects of pesticides. We used the zebrafish model to test the hypothesis that developmental exposure to low doses of the pyrethroid deltamethrin results in persistent alterations in dopaminergic gene expression, neurochemistry, and locomotor activity. Zebrafish embryos were treated with deltamethrin (0.25-0.50 µg/l), at concentrations below the LOAEL, during the embryonic period [3-72 h postfertilization (hpf)], after which transferred to fresh water until the larval stage (2-weeks postfertilization). Deltamethrin exposure resulted in decreased transcript levels of the D1 dopamine (DA) receptor (drd1) and increased levels of tyrosine hydroxylase at 72 hpf. The reduction in drd1 transcripts persisted to the larval stage and was associated with decreased D2 dopamine receptor transcripts. Larval fish, exposed developmentally to deltamethrin, had increased levels of homovanillic acid, a DA metabolite. Since the DA system is involved in locomotor activity, we measured the swim activity of larval fish following a transition to darkness. Developmental exposure to deltamethrin significantly increased larval swim activity which was attenuated by concomitant knockdown of the DA transporter. Acute exposure to methylphenidate, a DA transporter inhibitor, increased swim activity in control larva, while reducing swim activity in larva developmentally exposed to deltamethrin. Developmental exposure to deltamethrin causes locomotor deficits in larval zebrafish, which is likely mediated by dopaminergic dysfunction. This highlights the need to understand the persistent effects of low-dose neurotoxicant exposure during development.

Chronic exposure of killifish to a highly polluted environment desensitizes estrogen-responsive reproductive and biomarker genes.

Reproductive and endocrine disruption is commonly reported in aquatic species exposed to complex contaminant mixtures. We previously reported that Atlantic killifish (Fundulus heteroclitus) from the chronically contaminated Newark Bay, NJ, exhibit multiple endocrine disrupting effects, including inhibition of vitellogenesis (yolk protein synthesis) in females and false negative vitellogenin biomarker responses in males. Here, we characterized the effects on estrogen signaling and the transcriptional regulation of estrogen-responsive genes in this model population. First, a dose-response study tested the hypothesis that reproductive biomarkers (vtg1, vtg2, chg H, chg Hm, chg L) in Newark Bay killifish are relatively less sensitive to 17ß-estradiol at the transcriptional level, relative to a reference (Tuckerton, NJ) population. The second study assessed expression for various metabolism (cyp1a, cyp3a30, mdr) and estrogen receptor (ER α, ER ßa, ER ßb) genes under basal and estrogen treatment conditions in both populations. Hepatic metabolism of 17ß-estradiol was also evaluated in vitro as an integrated endpoint for adverse effects on metabolism. In the third study, gene methylation was evaluated for promoters of vtg1 (8 CpGs) and vtg2 (10 CpGs) in both populations, and vtg1 promoter sequences were examined for single nucleotide polymorphism (SNPs). Overall, these studies show that multi-chemical exposures at Newark Bay have desensitized all reproductive biomarkers tested to estrogen. For example, at 10ng/g 17ß-estradiol, inhibition of gene induction ranged from 62% to 97% for all genes tested in the Newark Bay population, relative to induction levels in the reference population. The basis for this recalcitrant phenotype could not be explained by a change in 17ß-estradiol metabolism, nuclear estrogen receptor expression, promoter methylation (gene silencing) or SNPs, all of which were unaltered and normal in the Newark Bay population. The decreased transcriptional sensitivity of estrogen-responsive genes is suggestive of a broad effect on estrogen receptor pathway signaling, and provides insight into the mechanisms of the endocrine disrupting effects in the Newark Bay population.

Manipulation of the HIF-Vegf pathway rescues methyl tert-butyl ether (MTBE)-induced vascular lesions.

Methyl tert-butyl ether (MTBE) has been shown to be specifically anti-angiogenic in piscine and mammalian model systems at concentrations that appear non-toxic in other organ systems. The mechanism by which MTBE targets developing vascular structures is unknown. A global transcriptome analysis of zebrafish embryos developmentally exposed to 0.00625-5mM MTBE suggested that hypoxia inducible factor (HIF)-regulated pathways were affected. HIF-driven angiogenesis via vascular endothelial growth factor (vegf) is essential to the developing vasculature of an embryo. Three rescue studies were designed to rescue MTBE-induced vascular lesions: pooled blood in the common cardinal vein (CCV), cranial hemorrhages (CH), and abnormal intersegmental vessels (ISV), and test the hypothesis that MTBE toxicity was HIF-Vegf dependent. First, zebrafish vegf-a over-expression via plasmid injection, resulted in significantly fewer CH and ISV lesions, 46 and 35% respectively, in embryos exposed to 10mM MTBE. Then HIF degradation was inhibited in two ways. Chemical rescue by N-oxaloylglycine significantly reduced CCV and CH lesions by 30 and 32% in 10mM exposed embryos, and ISV lesions were reduced 24% in 5mM exposed zebrafish. Finally, a morpholino designed to knock-down ubiquitin associated von Hippel-Lindau protein, significantly reduced CCV lesions by 35% in 10mM exposed embryos. In addition, expression of some angiogenesis related genes altered by MTBE exposure were rescued. These studies demonstrated that MTBE vascular toxicity is mediated by a down regulation of HIF-Vegf driven angiogenesis. The selective toxicity of MTBE toward developing vasculature makes it a potentially useful chemical in the designing of new drugs or in elucidating roles for specific angiogenic proteins in future studies of vascular development.

Inhibition of vitellogenin gene induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin is mediated by aryl hydrocarbon receptor 2 (AHR2) in zebrafish (Danio rerio).

Vitellogenins are hepatically derived yolk-protein precursors required for oogenesis in all oviparous teleosts. Altered gene-regulation of vitellogenesis by environmental contaminants can have profound effects on reproductive success, and ultimately population sustainability. To better understand chemical effects on vitellogenin gene regulation, we tested the hypothesis that activation of the aryl hydrocarbon receptor 2 (AHR2) by dioxin inhibits the estrogen receptor pathway regulation of 3 vitellogenin genes (vtg1-3) in vivo, using zebrafish (Danio rerio) as a model teleost. Using an embryo-larval bioassay, embryos were either treated with 1000 pptr (parts-per-trillion, pg/mL) 17α-ethynylestradiol (EE2) alone from 6h post fertilization (hpf) to 4 days post fertilization (dpf), or pre-treated with dioxin (4-5 hpf) prior to EE2. Pre-treatment with 400 pptr 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) or 1,2,3,7,8-pentachlorodibenzo-p-dioxin inhibited the EE2 induction of vtg1, vtg2 and vtg3 by >95% (p≤0.05). In comparison, a splice-blocking AHR2 morpholino used to down-regulate ahr2 expression significantly reduced the inhibition of vtg1, vtg2 and vtg3 by 400 pptr 2,3,7,8-TCDD (20.7-27.4% rescue). These studies demonstrate that 2,3,7,8-TCDD directly inhibits the vitellogenin pathway in vivo through activation of the AHR2. This work provides evidence for AHR2 dependent cross-talk inhibition of vitellogenin genes and offers insight into anti-estrogenic reproductive effects observed in oviparous species exposed to AHR agonist contaminants.

Craniofacial abnormalities and altered wnt and mmp mRNA expression in zebrafish embryos exposed to gasoline oxygenates ETBE and TAME.

Gasoline additives ethyl tert butyl ether (ETBE) and tertiary amyl methyl ether (TAME) are used world wide, but the consequence of developmental exposure to these hydrophilic chemicals is unknown for aquatic vertebrates. The effect of ETBE and TAME on zebrafish embryos was determined following OECD 212 guidelines, and their toxicity was compared to structurally related methyl tert-butyl ether (MTBE), which is known to target developing vasculature. LC50s for ETBE and TAME were 14 mM [95% CI=10-20] and 10 mM [CI=8-12.5], respectively. Both chemicals caused dose dependent developmental lesions (0.625-10 mM), which included pericardial edema, abnormal vascular development, whole body edema, and craniofacial abnormalities. The lesions were suggestive of a dysregulation of WNT ligands and matrix metalloproteinase (MMP) protein families based on their roles in development. Exposure to 5 mM ETBE significantly (p≤0.05) decreased relative mRNA transcript levels of mmp-9 and wnt3a, while 2.5 and 5 mM TAME significantly decreased wnt3a, and wnt8a. TAME also significantly decreased mmp-2 and -9 mRNA levels at 5mM. ETBE and TAME were less effective in altering the expression of vascular endothelial growth factor-a and -c, which were the only genes tested that were significantly decreased by MTBE. This is the first study to characterize the aquatic developmental toxicity following embryonic exposure to ETBE and TAME. Unlike MTBE, which specifically targets angiogenesis, ETBE and TAME disrupt multiple organ systems and significantly alter the mRNA transcript levels of genes required for general development.

Decreased vitellogenin inducibility and 17ß-estradiol levels correlated with reduced egg production in killifish (Fundulus heteroclitus) from Newark Bay, NJ.

Aquatic species inhabiting polluted estuaries are exposed to complex mixtures of xenobiotics which can alter normal reproduction. We previously reported that female Atlantic killifish (Fundulus heteroclitus) from the highly contaminated Newark Bay, NJ (USA) exhibited an inhibition of oocyte development due to reduced vitellogenin (egg-yolk precursor) levels. Our hypothesis was that the inhibition of oocyte development in Newark Bay killifish is due to (1) deficient levels of circulating 17ß-estradiol, and (2) a decreased sensitivity of the vitellogenin pathway to physiological doses of 17ß-estradiol. In the first study, adult naïve killifish from Tuckerton, NJ (reference) were caged at Tuckerton and Newark Bay. After 1 month, males caged at Newark Bay exhibited inductions of hepatic vitellogenin and estrogen receptor α, which were transient and returned to basal levels after 2 months (p≤0.05). In the second study, fecundity and 17ß-estradiol levels were measured in reproductively active adult females from Tuckerton and Newark Bay. Tuckerton females produced 140 eggs per female and Newark Bay females produced 11 eggs per female. Embryos from Newark Bay had 34% greater mortality and 28% less hatch, relative to Tuckerton. In addition, embryo mass and yolk-volume of Newark Bay embryos compared to Tuckerton embryos was 16% and 25% lower, respectively. Circulating 17ß-estradiol levels in Newark Bay females (0.26 ng/mL) were measured to be 8-fold lower than Tuckerton females (2.25 ng/mL). In the third study, adult killifish from both sites were dosed with 17ß-estradiol to assess the sensitivity of the vitellogenin pathway. At doses of 0.01, 0.1, 1 and 10 ng/g body weight, induction levels of circulating vitellogenin in Newark Bay males were significantly inhibited by 97, 99, 98 and 44%, respectively, compared to Tuckerton males. At doses of 0.01, 0.1, 1, 10 and 100 ng/g body weight, induction levels of circulating vitellogenin in Newark Bay females were inhibited by 89, 79, 61, 40 and 30%, respectively, compared to Tuckerton females. These differences in inducibility could not be explained by altered hepatic expression of estrogen receptors α, ßa or ßb. Based on the caged and dose-response studies, contaminants that down-regulate vitellogenin would interfere with its ability to be used as a biomarker for xeno-estrogen exposures. These studies demonstrate that contaminants within Newark Bay exert both estrogenic and anti-estrogenic responses which results in an overtly anti-estrogenic phenotype (reduced egg production due to inhibition of vitellogenesis).

Methyl tert butyl ether targets developing vasculature in zebrafish (Danio rerio) embryos.

Disruption of vascular endothelial growth factor (VEGF) signaling during early development results in abnormal angiogenesis and increased vascular lesions. Embryonic exposure to 0.625-10mM methyl tert butyl ether (MTBE), a highly water soluble gasoline additive, resulted in a dose dependent increase in pooled blood in the common cardinal vein (CCV), cranial hemorrhages and abnormal intersegmental vessels (ISVs). The EC50s for the lesions ranked in terms of likelihood to occur with MTBE exposure were: pooled blood in the CCV, 3.2 mM [95% CI: 2.2-4.7]>cranial hemorrhage, 11 mM [5.9-20.5]>abnormal ISV, 14.5 mM [6.5-32.4]. Organ systems other than the vascular system appear to develop normally, which suggests MTBE toxicity targets developing blood vessels. Equal molar concentrations (0.625-10mM) of the primary metabolites, tertiary butyl alcohol (TBA) and formaldehyde, did not result in vascular lesions, which suggested that the parent compound is responsible for the toxicity. Stage specific exposures were carried out to determine the developmental period most sensitive to MTBE vascular disruption. Embryos treated until 6-somites or treated after Prim-5 stages did not exhibit a significant increase in lesions, while embryos treated between 6-somites and Prim-5 had a significant increase in vascular lesions (p≤0.05). During the critical window for MTBE-induced vascular toxicity, expression of vegfa, vegfc, and flk1/kdr were significantly decreased 50, 70 and 40%, respectively. This is the first study to characterize disruption in vascular development following embryonic exposure to MTBE. The unique specificity of MTBE to disrupt angiogenesis may be mediated by the down regulation of critical genes in the VEGF pathway.

Microbially mediated O-methylation of bisphenol A results in metabolites with increased toxicity to the developing zebrafish (Danio rerio) embryo.

Bisphenol A (BPA) is used in the manufacture of plastics, and has been identified in various environmental matrices, including human serum and breast milk. The prevalence of BPA in the environment and the potential exposure to humans underscores the need to more fully understand the fate of BPA in the environment and the resulting effects and toxicity to humans and other organisms. Here we demonstrate that Mycobacterium species, including Mycobacterium vanbaalenii strain PYR-1, are able to O-methylate BPA to its mono- and dimethyl ether derivatives (BPA MME and BPA DME, respectively). The O-methylation of BPA results in metabolites with increased toxicity as shown from differences in survival and occurrence of developmental lesions in developing zebrafish embryos exposed to BPA, BPA MME, and BPA DME. The mono- and dimethyl ether derivatives were more toxic than BPA, resulting in increased mortality at 5 (LC(50) = 0.66 and 1.2 mg L(-1)) and 28 (LC(50) = 0.38, <0.5 mg L(-1)) days post fertilization. Furthermore, exposure to either of the O-methylated metabolites resulted in an increase in the incidence of developmental lesions as compared to BPA exposure. These data illustrate a new mechanism for microbial transformation of BPA, producing metabolites warranting further study to understand their prevalence and effects in the environment.

Embryonic exposure to tetrabromobisphenol A and its metabolites, bisphenol A and tetrabromobisphenol A dimethyl ether disrupts normal zebrafish (Danio rerio) development and matrix metalloproteinase expression.

Tetrabromobisphenol A (TBBPA) is a widely used brominated flame retardant that is persistent in the environment and detected in human serum and breast milk. TBBPA is microbiologically transformed in anaerobic environments to bisphenol A (BPA) and in aerobic environments to TBBPA dimethyl ether (TBBPA DME). Despite the detection of TBBPA DME in the environment, the resulting toxicity is not known. The relative toxicity of TBBPA, BPA and TBBPA DME was determined using embryonic exposure of zebrafish, with BPA and TBBPA DME exhibiting lower potency than TBBPA. TBBPA exposure resulted in 100% mortality at 3 (1.6mg/L) and 1.5µM (0.8mg/L), whereas BPA and TBBPA DME did not result in significant embryonic mortality in comparison to controls. While all three caused edema and hemorrhage, only TBBPA specifically caused decreased heart rate, edema of the trunk, and tail malformations. Matrix metalloproteinase (MMP) expression was measured due to the role of these enzymes in the remodeling of the extracellular matrix during tissue morphogenesis, wound healing and cell migration. MMP-2, -9 and -13 expression increased (2-8-fold) after TBBPA exposure followed by an increase in the degradation of collagen I and gelatin. TBBPA DME exposure resulted in only a slight increase (less than 2-fold) in MMP expression and did not significantly increase enzymatic activity. These data suggest that TBBPA is more potent than BPA or TBBPA DME and indicate that the trunk and tail phenotypes seen after TBBPA exposure could be due in part to alteration of proper MMP expression and activity.

Impaired reproductive health of killifish (Fundulus heteroclitus) inhabiting Newark Bay, NJ, a chronically contaminated estuary.

A battery of biomarkers were used to evaluate the reproductive health and contaminant exposure of Atlantic killifish (Fundulus heteroclitus) inhabiting the heavily industrialized Newark Bay and a reference population from Great Bay, Tuckerton, NJ. The biomarkers investigated included classical endpoints (gonad and liver histopathology, body and tissue morphometrics), hepatic mRNA expression (CYP1A and vitellogenin I), hepatic protein levels (CYP1A and vitellogenin), gonadal aromatase mRNA expression, and chemical exposure analyses (bile PAHs). Our data showed no significant differences between populations for body size and body weight. However, Newark Bay killifish exhibited molecular and morphological changes indicative of impaired reproductive health and endocrine disruption compared to the reference population. Newark Bay males had decreased gonad weight, altered testis development and decreased gonadal aromatase mRNA expression. Newark Bay females had decreased gonad weight, inhibited gonadal development, decreased hepatic vitellogenin production (mRNA and protein) and increased mRNA expression of gonadal aromatase. In addition, Newark Bay females had a significant increase in the percent of pre-vitellogenic follicles (43% at Tuckerton, 64% at Newark Bay) and a significantly decreased percent of follicles at the mid-vitellogenic and mature stages (25% mature at Tuckerton and 3% at Newark Bay). In addition to reproductive endpoints, killifish at Newark Bay exhibited high basal levels of CYP1A mRNA and protein expression which indicated exposure to aryl hydrocarbon receptor (AhR) agonists. An inverse relationship between hepatic CYP1A protein and hepatic vitellogenin mRNA expression was established suggesting a possible link between AhR agonist exposure and vitellogenesis. Killifish in the NY-NJ Harbor Estuary are exposed to a number of chemicals that can interact with the AhR pathway and stimulate enzymatic activity along with chemicals that can modify reproductive success in this indigenous species. Similar effects on the reproductive development in less resilient species may limit their ability to repopulate the NY-NJ Harbor Estuary and similarly contaminated water systems.

Developmental neurotoxicity of pyrethroid insecticides in zebrafish embryos.

Pyrethroid insecticides are one of the most commonly used residential and agricultural insecticides. Based on the increased use of pyrethroids and recent studies showing that pregnant women and children are exposed to pyrethroids, there are concerns over the potential for developmental neurotoxicity. However, there have been relatively few studies on the developmental neurotoxicity of pyrethroids. In this study, we sought to investigate the developmental toxicity of six common pyrethroids, three type I compounds (permethrin, resmethrin, and bifenthrin) and three type II compounds (deltamethrin, cypermethrin, and lambda-cyhalothrin), and to determine whether zebrafish embryos may be an appropriate model for studying the developmental neurotoxicity of pyrethroids. Exposure of zebrafish embryos to pyrethroids caused a dose-dependent increase in mortality and pericardial edema, with type II compounds being the most potent. At doses approaching the LC(50), permethrin and deltamethrin caused craniofacial abnormalities. These findings are consistent with mammalian studies demonstrating that pyrethroids are mildly teratogenic at very high doses. However, at lower doses, body axis curvature and spasms were observed, which were reminiscent of the classic syndromes observed with pyrethroid toxicity. Treatment with diazepam ameliorated the spasms, while treatment with the sodium channel antagonist MS-222 ameliorated both spasms and body curvature, suggesting that pyrethroid-induced neurotoxicity is similar in zebrafish and mammals. Taken in concert, these data suggest that zebrafish may be an appropriate alternative model to study the mechanism(s) responsible for the developmental neurotoxicity of pyrethroid insecticides and aid in identification of compounds that should be further tested in mammalian systems.

Glucocorticoids alter craniofacial development and increase expression and activity of matrix metalloproteinases in developing zebrafish (Danio rerio).

Teratogenic effects are observed following long-term administration of glucocorticoids, although short-term glucocorticoid therapy is still utilized to reduce fetal mortality, respiratory distress syndrome, and intraventricular hemorrhage in preterm infants. However, the mechanism of glucocorticoid-induced teratogenicity is unknown. We hypothesize that glucocorticoid-induced teratogenesis is mediated through the glucocorticoid receptor (GR) and results from altering the expression and activity of the matrix metalloproteinases (MMPs). During embryogenesis, degradation of the extracellular matrix to allow for proper cellular migration and tissue organization is a tightly regulated process requiring appropriate temporal and spatial expression and activity of the MMPs. Studies have demonstrated that MMP gene expression can be either inhibited or induced by glucocorticoids in a variety of model systems. Using the zebrafish (Danio rerio) as a model of development, the data presented here demonstrate that embryonic exposure to the glucocorticoids dexamethasone or hydrocortisone increased expression of two gelatinases, MMP-2 ( approximately 1.5-fold) and MMP-9 (7.6- to 9.0-fold), at 72 h postfertilization (hpf). Further, gelatinase activity was increased approximately threefold at 72 hpf following glucocorticoid treatment, and changes in craniofacial morphogenesis were also observed. Cotreatment of zebrafish embryos with each glucocorticoid and the GR antagonist RU486 resulted in attenuation of glucocorticoid-induced increases in MMP expression (52-84% decrease) and activity (41-94% decrease). Furthermore, the abnormal craniofacial phenotype observed following glucocorticoid exposure was less severe following RU486 cotreatment. These studies demonstrate that in the embryonic zebrafish, dexamethasone, and hydrocortisone alter expression and activity of MMP-2 and -9, and suggest that these increases may be mediated through the GR.

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by human CYP1B1 genetic variants.

Human cytochrome P450 1B1 (CYP1B1) plays a critical role in the metabolic activation of a variety of procarcinogens, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The existence of human CYP1B1 missense genetic variants has been demonstrated, but their activities in metabolizing PhIP are unknown. In this study, we expressed 15 naturally occurring CYP1B1 variants (with either single or multiple amino acid substitutions) and determined their activity changes in metabolizing PhIP to its two major metabolites, 2-hydroxyamino-PhIP and 4'-hydroxy-PhIP. Although the PhIP-metabolizing activities of four variants (Ala(119)Ser, Pro(379)Leu, Ala(443)Gly, Arg(48)Gly/Leu(432)Val) were comparable with that of the expressed wild-type CYP1B1, five variants (Trp(57)Cys, Gly(61)Glu, Arg(48)Gly/Ala(119)Ser, Arg(48)Gly/Ala(119)Ser/Leu(432)Val, Arg(48)Gly/Ala(119)Ser/Leu(432)Val/Ala(443)Gly) exhibited more than 2-fold decrease in activity and a reduction in the catalytic efficiency (V(max)/K(m)) for both N- and 4-hydroxylation of PhIP. Six variants (Gly(365)Trp, Glu(387)Lys, Arg(390)His, Pro(437)Leu, Asn(453)Ser, Arg(469)Trp) showed little activity in PhIP metabolism, but the molecular mechanisms involved are apparently different. The microsomal CYP1B1 protein level was significantly decreased for the Trp(365), Lys(387), and His(390) variants and was not detectable for the Ser(453) variant. In contrast, there was no difference between the Trp(469) variant and the wild-type in the microsomal CYP1B1 protein level and P450 content but the Trp(469) variant totally lost its metabolic activity toward PhIP. The Leu(437) variant also had a substantial amount of CYP1B1 protein in the microsomes, but there was a lack of detectable P450 peak and activity. Our results should be useful in selecting appropriate CYP1B1 variants as cancer susceptibility biomarkers for human population studies related to PhIP exposure.

The intersection between the aryl hydrocarbon receptor (AhR)- and retinoic acid-signaling pathways.

Data from a variety of animal and cell culture model systems have demonstrated an interaction between the aryl hydrocarbon receptor (AhR)- and retinoic acid (RA)-signaling pathways. The AhR(1) was originally identified as the receptor for the polycyclic aromatic hydrocarbon family of environmental contaminants; however, recent data indicate that the AhR binds to a variety of endogenous and exogenous compounds, including some synthetic retinoids. In addition, activation of the AhR pathway alters the function of nuclear hormone-signaling pathways, including the estrogen, thyroid, and RA pathways. Activation of the AhR pathway through exposure to environmental compounds results in significant changes in RA synthesis, catabolism, transport, and excretion. Some effects on retinoid homeostasis mediated by the AhR pathway may result from the interactions of these two pathways at the level of activating or repressing the expression of specific genes. This chapter will review these two pathways, the evidence demonstrating a link between them, and the data indicating the molecular basis of the interactions between these two pathways.

The impact of aryl hydrocarbon receptor signaling on matrix metabolism: implications for development and disease.

The aryl hydrocarbon receptor (AhR) was identified as the receptor for polycyclic aromatic hydrocarbons and related compounds. However, novel data indicate that the AhR binds a variety of unrelated endogenous and exogenous compounds. Although AhR knockout mice demonstrate that this receptor has a role in normal development and physiology, the function of this receptor is still unclear. Recent evidence suggests that AhR signaling also alters the expression of genes involved in matrix metabolism, specifically the matrix metalloproteinases (MMPs). MMP expression and activity is critical to normal physiological processes that require tissue remodeling, as well as in mediating the progression of a variety of diseases. MMPs not only degrade structural proteins, but are also important mediators of cell signaling near or at the cell membrane through exposure of cryptic sites, release of growth factors, and cleavage of receptors. Therefore, AhR modulation of MMP expression and activity may be critical, not only in pathogenesis, but also in understanding the endogenous function of the AhR. In this review we will examine the data indicating a role for the AhR-signaling pathway in the regulation of matrix remodeling, and discuss potential molecular mechanisms.

Interaction between the aryl hydrocarbon receptor and retinoic acid pathways increases matrix metalloproteinase-1 expression in keratinocytes.

Exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in a variety of pathological lesions in humans via activation of the aryl hydrocarbon receptor (AhR) pathway. It has become apparent that this pathway interacts with a variety of signaling pathways that are believed to be involved in mediating TCDD/AhR biological effects. Our hypothesis is that TCDD mediates these pathological lesions by directly altering the expression of genes involved in matrix deposition and remodeling and that the retinoic acid signaling pathway is involved in modulating TCDD-induced effects. Therefore, we examined the effect of TCDD and all-trans retinoic acid (atRA) on the expression of matrix metalloproteinase-1 (MMP-1, interstitial collagenase), one of the proteolytic enzymes that degrade type I collagen, in normal human keratinocytes. The data show that TCDD exposure results in increased MMP-1 expression in keratinocytes that is further enhanced by co-treatment with all-trans retinoic acid. TCDD-induced expression of MMP-1 appears to be mediated through two AP-1 elements in the proximal promoter of the MMP-1 gene. However, retinoic acid-mediated induction of keratinocyte MMP-1 is a result of both promoter activation and increased mRNA stability. These findings are the first to demonstrate TCDD-induced expression of MMP-1 and to demonstrate interactions between the TCDD/AhR and retinoic acid pathways on MMP-1 expression.