Phe Toxicity Research Articles

Volumetric brain reductions in adult patients with phenylketonuria and their relationship with blood phenylalanine levels

BackgroundContinued dietary treatment since early diagnosis through newborn screening programs usually prevents brain-related complications in phenylketonuria (PKU). However, subtle neurocognitive and brain alterations may be observed in some adult patients despite early treatment. Nevertheless, neuropsychological and neuroimaging studies in the field remain scarce.ObjectivesThis work aimed to determine possible neuropsychological and structural brain alterations in treated adult patients with PKU.MethodsThirty-five patients with PKU and 22 healthy controls (HC) underwent neuropsychological assessment and T1-weighted magnetic resonance imaging on a 3 T scanner. FreeSurfer (v.7.1) was used to obtain volumetric measures and SPSS (v27.0.1.0) was used to analyze sociodemographic, neuropsychological, volumetric, and clinical data (p < 0.05).ResultsAdult patients with PKU showed significantly lower performance than HC in Full Scale IQ (t = 2.67; p = .010) from the WAIS-IV. The PKU group also showed significantly lower volumes than HC in the pallidum (U = 224.000; p = .008), hippocampus (U = 243.000; p = .020), amygdala (U = 200.000; p = .002), and brainstem (t = 3.17; p = .006) as well as in total cerebral white matter volume (U = 175.000; p = .001). Blood phenylalanine (Phe) levels in PKU patients were negatively correlated with the pallidum (r = -0.417; p = .013) and brainstem (r = -0.455, p = .006) volumes.ConclusionsAdult patients with early-treated PKU showed significantly lower global intelligence than HC. Moreover, these patients showed reduced global white matter volume as well as reductions in the volume of several subcortical grey matter structures, which might be related to the existence of underlying neurodevelopmental alterations. Higher blood Phe levels were also negatively correlated with pallidum and brainstem, suggesting a higher vulnerability of these structures to Phe toxicity.

The combined effects of phenanthrene and micro-/nanoplastics mixtures on the cellular stress responses of the thick-shell mussel Mytilus coruscus

Pollution with complex mixtures of contaminants including micro- and nano-plastics (MNPs) and organic pollutants like polycyclic aromatic hydrocarbons (PAH) poses a major threat to coastal marine ecosystems. Toxic mechanisms of contaminant mixtures are not well understood in marine organisms. We studied the effects of single and combined exposures to polycyclic aromatic hydrocarbon phenanthrene (Phe) and MNPs mixture with sizes of 70 nm, 5 μm and 100 μm on the immune health and oxidative stress parameters in the thick-shell mussel Mytilus coruscus. Immune cells (hemocytes) were more sensitive to the pollutant-induced oxidative stress than the gills. In hemocytes of co-exposed mussels, elevated mortality, lower lysosomal content, high production of reactive oxygen species (ROS) and decrease mitochondrial were found. Disparate responses of antioxidant enzymes in the hemolymph (e.g. increased superoxide dismutase (SOD) activity without a corresponding increase in catalase (CAT) in Phe exposures and an increase in CAT without a change in SOD in MNPs exposures) suggests misbalance of the antioxidant defense in the pollutant-exposed mussels. Gill lacked pronounced oxidative stress response showing a decline in ROS and antioxidant levels. Tissue-specific single and combined effects of Phe and MNPs suggest variation in bioavailability and/or different sensitivity to these pollutants in the studied tissues. Notably, the combined effects of MNPs and Phe were additive or antagonistic, showing that MNPs do not enhance and occasionally mitigate the toxic effects of Phe on the hemocytes and the gills of the mussels. Overall, our study sheds light on the impact of long-term exposure to MNPs and Phe mixtures on mussels, showing high sensitivity of the immune system and modulation of the Phe toxicity by MNPs co-exposure. These findings that may have implications for understanding the impacts of combined PAH and MNPs pollution on the health of mussel populations from polluted coastal habitats.

Cardiac toxicity of phenanthrene depends on developmental stage in Atlantic cod (Gadus morhua)

Complex mixtures like crude oil, and single components such as Phenanthrene (Phe), induce cardiotoxicity by interfering with excitation-contraction coupling. However, recent work has demonstrated that the timing of pollutant exposure during embryogenesis greatly impacts the degree of cardiac dysfunction caused. Here, we aimed to clarify the temporal dependence of Phe toxicity and the downstream effects of cardiac dysfunction using Atlantic cod (Gadus morhua). Phe (nominal concentration, 1.12 μmol/L), or the L-type‑calcium channel blocker Nicardipine (Nic) (nominal concentration, 2 and 4 μmol/L), were individually applied to cod embryos either during cardiogenesis (early) or after the onset of cardiac function (late). Phe toxicity was highly dependent on the timing of exposure. Exposure after the onset of cardiac function (i.e. late) caused more severe cardiac and extracardiac abnormalities at 3 days post hatching (dph) than early exposure. Late Phe exposure resulted in a smaller ventricle, eliminated ventricular contraction, and reduced atrial contraction. In contrast, early Phe exposure did not have an effect on cardiac development and function. This temporal difference was not as evident in the Nic treatment. Early Nic exposure created similar morphological phenotypes to the late Phe exposure. The two treatments (early Nic and late Phe) also shared a cardiofunctional phenotype, comprised of eliminated ventricular, and reduced atrial, contraction. These data suggest that extracardiac abnormalities, such as the craniofacial deformities seen after late embryonic exposure to cardiotoxic oil components and mixtures, are mostly downstream effects of cardiac dysfunction.

Hibiscus sabdariffa Extract Protects HaCaT Cells against Phenanthrene-Induced Toxicity through the Regulation of Constitutive Androstane Receptor/Pregnane X Receptor Pathway.

Phenanthrene (Phe) exposure is associated with skin ageing, cardiotoxicity and developmental defects. Here, we investigated the mode of Phe toxicity in human keratinocytes (HaCaT cells) and the attenuation of toxicity on pre-treatment (6 h) with ethanol extract of Hibiscus sabdariffa calyxes (HS). Cell viability, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm) alteration, changes in the transcriptional activity of selected genes involved in phase I and II metabolism, antioxidant response and gluconeogenesis, western blot and docking studies were performed to determine the protective effect of HS against Phe. Phe (250 μM) induced cytotoxicity in HaCaT cells through AhR-independent, CAR/PXR/RXR-mediated activation of CYP1A1 and the subsequent alterations in phase I and II metabolism genes. Further, CYP1A1 activation by Phe induced ROS generation, reduced ΔΨm and modulated antioxidant response, phase II metabolism and gluconeogenesis-related gene expression. However, pre-treatment with HS extract restored the pathological changes observed upon Phe exposure through CYP1A1 inhibition. Docking studies showed the site-specific activation of PXR and CAR by Phe and inhibition of CYP1A1 and CYP3A4 by the bioactive compounds of HS similar to that of the positive controls tested. Our results conclude that HS extract can attenuate Phe-induced toxicity in HaCaT cells through CAR/PXR/RXR mediated inhibition of CYP1A1.

Distribution of phenanthrene in the ospho2 reveals the involvement of phosphate on phenanthrene translocation and accumulation in rice

The intricate mechanisms involved in the acquisition and translocation of polycyclic aromatic hydrocarbons (PAHs) in plants have not been elucidated. Phosphate (Pi) is the bioavailable form of essential macronutrient phosphorus, which is acquired and subsequently assimilated for plant optimal growth and development. Rice phosphate overaccumulator 2 (OsPHO2) is a central constituent of the regulation of Pi homeostasis in rice. In the present study, the role of OsPHO2 in regulating the translocation and accumulation of phenanthrene (Phe) and the involvement of Pi in this process were investigated. The temporal study (1 d-35 d) revealed a significant and gradual increase of Phe accumulation in Pi-deprived roots of wild-type (WT) seedlings. Compared with the WT, the concentrations of Phe were significantly higher in the shoots of ospho2 (OsPHO2 mutant) grown hydroponically with Phe (1.5 mg/L) under +Pi (200 μM) and -Pi (10 μM) conditions. The sap experiment clearly showed the significant increases in levels of Phe in the xylem sap of ospho2 than the WT grown hydroponically with Phe and +Pi. Further, the concentrations of both Phe and P were coordinately higher in the culms and flag leaves of the mutants than WT at maturity in potting soil with LPhe (6 mg/kg) and HPhe (60 mg/kg). However, the concentrations of Phe in the seeds were comparable in the WT and mutants, suggesting a pivotal of OsPHO2 in attenuating Phe toxicity in the seed. In +Phe WT, the relative expression level of OsPHO2 in the shoots was significantly lower, while those of Pi transporters (PTs) OsPT4 and OsPT8 were significantly higher in the roots compared with -Phe. Together, the results provided evidence towards the involvement of Pi in OsPHO2-regulated translocation and accumulation of Phe in rice.

Microplastics increase the accumulation of phenanthrene in the ovaries of marine medaka (Oryzias melastigma) and its transgenerational toxicity

Microplastics (MPs) are considered to exacerbate the impacts of hydrophobic organic pollutants on aquatic organisms through the carrier function, but whether MPs affect the transgenerational toxicity of pollutants is unclear. This study exposed adult female marine medaka to phenanthrene (Phe)-adsorbed MPs for 60 days to investigate the effects of MPs on the bioaccumulation, reproductive and transgenerational toxicity of Phe. Compared to Phe alone, co-exposure to Phe and 200 μg/L MPs significantly increased Phe bioaccumulation in the intestines and ovaries. Phe alone and Phe combined with MPs disrupted the regulation of the hypothalamus-pituitary-gonadal axis, and reduced vitellogenin levels and reproductive capacity of female fish. In particular, co-exposure to 200 μg/L MPs and Phe increased the rate of follicular atresia, inhibited ovarian maturity, and aggravated reproductive toxicity. Notably, maternal uptake of Phe could be transferred to the offspring, and embryonic accumulation increased with the concentrations of MPs. Moreover, MPs aggravated Phe-induced bradycardia in embryos, suggesting that MPs exacerbated the transgenerational toxicity of Phe. These findings reveal that the growing number of MPs in the ocean might amplify the adverse effects of organic pollutants on the health and population stability of marine fishes, and this problem merits more attention.

Long-term exposure to environmental levels of phenanthrene disrupts spermatogenesis in male mice

Phenanthrene (Phe) is a tricyclic polycyclic aromatic hydrocarbon with high bioavailability under natural exposure. However, there are few studies on the reproductive toxicity of Phe in mammals. In this study, male Kunming mice were gavaged once every two days with Phe (5, 50, and 500 ng/kg) for 28 weeks. The accumulation levels of Phe in the testis were dose-dependently increased. Histopathological staining showed that Phe exposure reduced the number of spermatogonia, sperm and Sertoli cells. The percentage of testicular apoptotic cells was significantly increased, which was further verified by the upregulated BAX protein. The expression of the GDNF/PI3K/AKT signaling pathway was downregulated, which might suppress the self-renewal and differentiation of spermatogonial stem cells. Meanwhile, Phe exposure inhibited the expression of Sertoli cell markers (Fshr, WT1, Sox9) and the Leydig cell marker Cyp11a1, indicating damage to the function of Sertoli cells and Leydig cells. Serum estrogen and testicular estrogen receptor alpha were significantly upregulated, while androgen receptor expression was downregulated. These alterations might be responsible for impaired spermatogenesis. This study provides new insights for evaluating the reproductive toxicity and potential mechanisms of Phe in mammals.

Transcriptome changes of Takifugu obscurus liver after acute exposure to phenanthrene.

Phenanthrene (Phe) is a model compound in polycyclic aromatic hydrocarbon (PAH) research. Reportedly, Phe treatment induced oxidative stress and histological disorders to Takifugu obscurus liver. In this study, to further explore the molecular responses of T. obscurus liver to Phe exposure, transcriptome sequencing was applied to compare mRNA transcription profiles between Phe treatment and the control. Compared with the control, 1,581 and 1,428 genes were significantly upregulated and downregulated in Phe treatment, respectively. Further analysis revealed that Phe treatment mainly upregulated genes in Ras-MAPK and PI3K-akt signaling pathways, which represented insulin resistance and further activated the FOXO signaling pathway. The triacylglycerol biosynthesis was promoted but the gluconeogenesis process was inhibited in response to Phe treatment, demonstrating that Phe exposure disturbed the sugar and lipid metabolism. Moreover, Phe treatment upregulated the Apelin-APJ and ErbB signaling pathways, promoting angiogenesis in T. obscurus liver. Insulin resistance, promoted triacylglycerol biosynthesis, and angiogenesis might explain the molecular mechanisms underlying carcinogenic toxicity of Phe. Overall, this study provides new insights to understand the environmental risk of Phe to fishes.

TiO2 nanoparticles enhanced bioaccumulation and toxic performance of PAHs via trophic transfer

Engineering nanoparticles (NPs) could act as accumulator and carrier of co-contaminants, affecting their fate and toxicity in environments. However, the effects of NPs on the bioaccumulation and trophic transfer of co-contaminants through the food chain and the ensuing effects on higher predators are unclear. In the present study, we investigated the effects of titanium dioxide nanoparticles (nTiO2) on the trophic transfer of phenanthrene (Phe) from prey Artemia salina to predator Scophthalmus maximus. We also evaluated the ensuing toxic performance of Phe in S. maximus after been transferred from A. salina in the presence and absence of nTiO2. The presence of nTiO2 significantly (p < 0.05) increased Phe accumulation in A. salina with higher bioconcentration factor (BCF) up to 90.9 than that of 38.6 in Phe exposure along. After trophic transfer, nTiO2 (1 mg/L) also promoted the bioaccumulation of Phe (1 μg/L) in predator S. maximus from 4.17 mg/kg to 7.85 mg/kg (dry weight). However, nTiO2 did not enhance the trophic transfer of Phe from A. salina to S. maximus since the biological magnification factor (BMF) decreased from 0.13 to 0.08. Nevertheless, the nTiO2-enhanced bioaccumulation of Phe did enhance Phe toxicity performance in predator S. maximus after trophic transfer, showing significant (p < 0.05) growth inhibition and changes of nutrient status in the predator, compared to those of the control. Further physio-biochemical investigations suggested that oxidative stress and inhibition of digestive functions might explain the growth inhibition in treatment with nTiO2 + Phe. This study demonstrates the first evidence that NP-enhanced bioaccumulation and toxic performance of co-existing pollutants across trophic transfer, which poses potential risks to marine ecosystems, and ultimately human health by seafood consumption.

Reducing phenanthrene uptake and translocation, and accumulation in the seeds by overexpressing OsNRT2.3b in rice

The uptake and accumulation of polycyclic aromatic hydrocarbons (PAHs) in crops have gained much attention due to their toxicity to humans. Nitrogen (N) is an essential element for plant growth and has also been implicated in the acquisition and acropetal translocation of PAHs. OsNRT2.3b encodes a nitrate (NO3−) transporter that is involved in the acquisition and mobilization of N in rice. Here, we investigated whether overexpression of OsNRT2.3b would exert any mitigating influence on the uptake and translocation of phenanthrene (Phe, a model PAH) in transgenic rice (Oryza sativa). The wild-type seedlings exhibited a reduction in plant height, primary root length, and shoot biomass when grown hydroponically in a medium supplemented with Phe. Acquisition of Phe by the roots and its subsequent translocation to shoots increased concomitantly with an increase in Phe concentration in the medium and duration of the treatment. OsNRT2.3b-overexpressing lines (Ox-6 and Ox-8) were generated independently. Compared with the wild-type, the concentration of Phe in Ox-6 and Ox-8 were significantly lower in the roots (47%–54%) and shoots (22%–31%) grown hydroponically with Phe (1 mg/L). Further, the wild-type and Ox lines were grown to maturity in a pot soil under Phe conditions and the concentrations of Phe and total N were assayed in the culms and flag leaves. Compared with the wild-type, in Ox lines the concentration of total N significantly increased in the culms (288%–366%) and flag leaves (12%–25%), while that of Phe significantly reduced in the culms (25%–28%) and flag leaves (18%–21%). The results revealed an antagonistic correlation between the concentration of total N and Phe. The concentration of Phe was also significantly lower (29%–38%) in the seeds of Ox lines than the wild-type. The study highlighted the efficacy of overexpressing OsNRT2.3b in mitigating the Phe toxicity by attenuating its acquisition, mobilization, and allocation to the seeds.

Low level of polystyrene microplastics decreases early developmental toxicity of phenanthrene on marine medaka (Oryzias melastigma)

Microplastics (MPs) have become global environmental concern. However, the effects of environmental concentrations of MPs, singly or in combination with organic pollutants, on the early development of marine fish remain unclear. In this study, fertilized eggs of marine medaka (Oryzias melastigma) were exposed to polystyrene MPs (0, 2, 20, 200 μg/L) and/or phenanthrene (Phe, 50 μg/L) for 28 days. The results revealed that MPs were accumulated on the chorion and ingested by larvae from 2 days post-hatching. High levels of MPs (20 and 200 μg/L) decreased the hatchability, delayed the hatching time, and suppressed the growth, whereas Phe inhibited hatching and caused malformations in larvae. The presence of MPs at 20 and 200 μg/L did not alter the toxicity of Phe. By contrast, combined exposure to 2 μg/L MPs and Phe increased the hatchability by 25.8%, decreased malformation and mortality rates, and restored Phe-induced abnormal expressions of cardiac development-related genes. The reduced early developmental toxicity could be attributed to the decreased bioavailability and bioaccumulation of Phe by the low level of MPs. These findings contradicted the view that MPs would aggravate the toxicity of organic pollutants, and future studies are warranted to elucidate the ecological risks of marine MPs.

A bone mineralization defect in the Pahenu2 model of classical phenylketonuria involves compromised mesenchymal stem cell differentiation

Osteopenia is observed in some patients affected by phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU). Bone density studies, in diverse PKU patient cohorts, have demonstrated bone disease is neither fully penetrant nor uniform in bone density loss. Biochemical assessment has generated a muddled perspective regarding mechanisms of the PKU bone phenotype where the participation of hyperphenylalaninemia remains unresolved. Osteopenia is realized in the Pahenu2 mouse model of classical PKU; although, characterization is incomplete. We characterized the Pahenu2 bone phenotype and assessed the effect of hyperphenylalaninemia on bone differentiation. Employing Pahenu2 and control animals, cytology, static and dynamic histomorphometry, and biochemistry were applied to further characterize the bone phenotype. These investigations demonstrate Pahenu2 bone density is decreased 33% relative to C57BL/6; bone volume/total volume was similarly decreased; trabecular thickness was unchanged while increased trabecular spacing was observed. Dynamic histomorphometry demonstrated a 25% decrease in mineral apposition. Biochemically, control and PKU animals have similar plasma cortisol, adrenocorticotropic hormone, and 25-hydroxyvitamin D. PKU animals show moderately increased plasma parathyroid hormone while plasma calcium and phosphate are reduced. These data are consistent with a mineralization defect. The effect of hyperphenylalaninemia on bone maturation was assessed in vitro employing bone-derived mesenchymal stem cells (MSCs) and their differentiation into bone. Using standard culture conditions, PAH deficient MSCs differentiate into bone as assessed by in situ alkaline phosphatase activity and mineral staining. However, PAH deficient MSCs cultured in 1200 μM PHE (metric defining classical PKU) show significantly reduced mineralization. These data are the first biological evidence demonstrating a negative impact of hyperphenylalaninemia upon bone maturation. In PAH deficient MSCs, expression of Col1A1 and Rankl are suppressed by hyperphenylalaninemia consistent with reduced bone formation and bone turnover. Osteopenia is intrinsic to PKU pathology in untreated Pahenu2 animals and our data suggests PHE toxicity participates by inhibiting mineralization in the course of MSC bone differentiation.

Graphene oxide in the marine environment: Toxicity to Artemia salina with and without the presence of Phe and Cd2+

Given the increasing potential of graphene oxide entering marine environments, it is imperative to assess the risks of GO on marine ecosystem, including its direct toxicity to marine organisms and indirect toxicity brought by co-existing aquatic pollutants, as a result of the remarkable adsorption capacity of GO. In the present study, the acute toxicity of GO, Phe, Cd2+, GO-Phe, and GO-Cd2+ to Artemia salina were systemically assessed and compared for the first time. Although the lethal effects of GO alone to A. salina only appeared at high GO dose (500 mg/L), its sublethal toxicity (growth inhibition) at concentrations as low as 1 mg/L was observed by microscopy, which was likely closely related to the GO-induced oxidative stress in A. salina. Compared with the toxicity of Phe alone, GO-Phe exhibited a synergistic effect to A. salina at a high GO concentration. For GO-Cd2+, the toxicity was positively correlated with both GO dose and Cd2+ dose. The increased toxicity of GO-Phe or GO-Cd2+ at high doses might be attributed to the promoted bioaccumulation of toxicants by GO, as the adhesion of GO complexes to intestinal tract of A. salina was observed during the toxicity tests, which probably resulted in further toxicological effects.

TiO2 nanoparticles in the marine environment: Impact on the toxicity of phenanthrene and Cd2 + to marine zooplankton Artemia salina

The impact of manufactured nanoparticles on the toxicity of co-existing pollutants in aquatic environments has raised increasing concerns. However, the toxicity of polycyclic aromatic hydrocarbons or metal ions in the presence of titanium dioxide nanoparticles (nTiO2) to marine zooplankton has been rarely reported. In the present study, the impacts of nTiO2 on the toxicity of phenanthrene (Phe) and cadium (Cd2+) to Artemia salina, a model marine zooplankton, were investigated. Although nTiO2 alone exerted a limited toxicity to A. salina within 48h of exposure, nTiO2 strongly altered the toxicity of Phe and Cd2+ to A. salina. Compared with the individual toxicities of pollutants to A. salina, the toxicities of Phe and Cd2+ increased by 2.0% and 12.2%, respectively, in the presence of 5mg/L nTiO2, but decreased by 24.5% and 57.1%, respectively, in the presence of 400mg/L nTiO2. These concentration-dependent impacts of nTiO2 on the toxicity of Phe or Cd2+ might be attributed to the concurrent functions of several interrelated factors including the adsorption of pollutants on nTiO2, the nTiO2-faciliated bioaccumulation of pollutants, the limited gut volume in organisms, and the aggregation and sedimentation behaviors of nTiO2. These results presented in the study could help understand the effects of manufactured nanomaterials in marine environments.

Assessment of immune status of yellowfin seabream (Acanthopagrus latus) during short term exposure to phenanthrene

The aim of the present investigation was to assess the immune status in yellowfin seabream (Acanthopagrus latus) exposed to different concentrations of phenanthrene (Phe) for 14days. In addition, the Phe accumulation in the fish muscle was measured during the experiment. Fish were injected with different concentrations (0, 2, 20 and 40mg/kg) of Phe and samples were taken from tissue and blood of fish 1, 4, 7 and 14days after injection. Exposure of fish to Phe caused a significant decrease in white blood cells, C3 and C4 levels, lysosomal membrane stability, lysozyme activity after 4days and antibacterial activity after 7days of the experiment. In contrast, cortisol level significantly increased after 4days. The concentration of Phe in fish muscle increased rapidly after 4days. The main tissue changes observed in the head kidney including increase in melanomacrophage centers (MMCs), empty spaces between cells and hemorrhage. The degree of tissue changes ranged from normal to moderate in Phe-treated fish. The size and number of MMCs in treated fish were significantly higher than control. In conclusion, Phe toxicity in yellowfin seabream can induce increased cortisol level, tissue changes and immune suppression.

Influence of temperature on phenanthrene toxicity towards nitrifying bacteria in three soils with different properties

This study focused on the combined effect of environmental conditions (temperature) and contamination (polycyclic aromatic hydrocarbons, PAHs) on the activity of soil microorganisms (nitrifying bacteria). Phenanthrene (Phe) at five contamination levels (0, 1, 10, 100 and 1000 mg kg−1 dry mass of soil) was employed as a model PAH compound in laboratory experiments that were conducted at three temperatures (i.e., 20 °C (recommended by ISO 15685 method), 15 and 30 °C). Three soils with different properties were used in these studies, and the activity of the nitrifying bacteria was assessed based on nitrification potential (NP) determinations. For the statistical evaluation of the results, the ANCOVA (analysis of covariance) method for three independent variables (i.e., temperature, phenanthrene concentration, soil matrix (as a qualitative variable)) and their interactions was employed. The results indicated on the significant interaction of all studied factors. Temperature influenced the toxicity of Phe towards NP, and this effect was related to the Phe concentration as well as was varied for the different soils. A low content of soil organic matter (controlling bioavailability of phenanthrene to soil microorganisms) enhanced the combined effect of temperature and Phe toxicity, and a high biological activity of the soil (high NP values) increased the effect of high temperature on the Phe stimulatory influence. The results indicate that the temperature should not be neglected in tests evaluating PAH ecotoxicity, especially for reliable ecological risk assessment.

DNA methylation in the pathophysiology of hyperphenylalaninemia in the PAH(enu2) mouse model of phenylketonuria.

Phenylalanine hydroxylase deficient phenylketonuria (PKU) is the paradigm for a treatable inborn error of metabolism where maintaining plasma phenylalanine (Phe) in the therapeutic range relates to improved clinical outcomes. While Phe is the presumed intoxicating analyte causal in neurologic damage, the mechanism(s) of Phe toxicity has remained elusive. Altered DNA methylation is a recognized response associated with exposure to numerous small molecule toxic agents. Paralleling this effect, we hypothesized that chronic Phe over-exposure in the brain would lead to aberrant DNA methylation with secondary influence upon gene regulation that would ultimately contribute to PKU neuropathology. The PAH(enu2) mouse models human PKU with intrinsic hyperphenylalaninemia, abnormal response to Phe challenge, and neurologic deficit. To examine this hypothesis, we assessed DNA methylation patterns in brain tissues using methylated DNA immunoprecipitation and paired end sequencing in adult PAH(enu2) animals maintained under either continuous dietary Phe restriction or chronic hyperphenylalaninemia. Heterozygous PAH(enu2/WT) litter mates served as controls for normal Phe exposure. Extensive repatterning of DNA methylation was observed in brain tissue of hyperphenylalaninemic animals while Phe restricted animals displayed an attenuated pattern of aberrant DNA methylation. Affected gene coding regions displayed aberrant hypermethylation and hypomethylation. Gene body methylation of noncoding RNA genes was observed and among these microRNA genes were prominent. Of particular note, observed only in hyperphenylalaninemic animals, was hypomethylation of miRNA genes within the imprinted Dlk1-Dio3 locus on chromosome 12. Aberrant methylation of microRNA genes influenced their expression which has secondary effects upon the expression of targeted protein coding genes. Differential hypermethylation of gene promoters was exclusive to hyperphenylalaninemic PAH(enu2) animals. Genes with synaptic involvement were targets of promoter hypermethylation that resulted in down-regulation of their expression. Gene dysregulation secondary to abnormal DNA methylation may be contributing to PKU neuropathology. These results suggest drugs that prevent or correct aberrant DNA methylation may offer a novel therapeutic option to management of neurological symptoms in PKU patients.

The Fas/Fas Ligand Death Receptor Pathway Contributes to Phenylalanine-Induced Apoptosis in Cortical Neurons

Phenylketonuria (PKU), an autosomal recessive disorder of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene, leads to childhood mental retardation by exposing neurons to cytotoxic levels of phenylalanine (Phe). A recent study showed that the mitochondria-mediated (intrinsic) apoptotic pathway is involved in Phe-induced apoptosis in cultured cortical neurons, but it is not known if the death receptor (extrinsic) apoptotic pathway and endoplasmic reticulum (ER) stress-associated apoptosis also contribute to neurodegeneration in PKU. To answer this question, we used specific inhibitors to block each apoptotic pathway in cortical neurons under neurotoxic levels of Phe. The caspase-8 inhibitor Z-IETD-FMK strongly attenuated apoptosis in Phe-treated neurons (0.9 mM, 18 h), suggesting involvement of the Fas receptor (FasR)-mediated cell death receptor pathway in Phe toxicity. In addition, Phe significantly increased cell surface Fas expression and formation of the Fas/FasL complex. Blocking Fas/FasL signaling using an anti-Fas antibody markedly inhibited apoptosis caused by Phe. In contrast, blocking the ER stress-induced cell death pathway with salubrinal had no effect on apoptosis in Phe-treated cortical neurons. These experiments demonstrate that the Fas death receptor pathway contributes to Phe-induced apoptosis and suggest that inhibition of the death receptor pathway may be a novel target for neuroprotection in PKU patients.

Characterization of a Fungal Maleylacetoacetate Isomerase Gene and Identification of Its Human Homologue

We have previously used Aspergillus nidulans as a fungal model for human phenylalanine catabolism. This model was crucial for our characterization of the human gene involved in alcaptonuria. We use here an identical approach to characterize at the cDNA level the human gene for maleylacetoacetate isomerase (MAAI, EC 5.2.1.2), the only as yet unidentified structural gene of the phenylalanine catabolic pathway. We report here the first characterization of a gene encoding a MAAI enzyme from any organism, the A. nidulans maiA gene. maiA disruption prevents growth on phenylalanine (Phe) and phenylacetate and results in the absence of MAAI activity in vitro and Phe toxicity. The MaiA protein shows strong amino acid sequence identity to glutathione S-transferases and has MAAI activity when expressed in Escherichia coli. maiA is clustered with fahA and hmgA, the genes encoding the two other enzymes of the common part of the Phe/phenylacetate pathways. Based on the high amino acid sequence conservation existing between other homologous A. nidulans and human enzymes of this pathway, we used the MaiA sequence in data base searches to identify human expressed sequence tags encoding its putative homologues. Four such cDNAs were sequenced and shown to be encoded by the same gene. They encode a protein with 45% sequence identity to MaiA, which showed MAAI activity when expressed in E. coli. Human MAAI deficiency would presumably cause tyrosinemia that would be characterized by the absence of succinylacetone, the diagnostic compound resulting from fumarylacetoacetate hydrolase deficiency in humans and fungi. Culture supernatants of an A. nidulans strain disrupted for maiA are succinylacetone-negative but specifically contain cis and/or trans isomers of 2, 4-dioxohept-2-enoic acid. We suggest that this compound(s) might be diagnostic for human MAAI deficiency.