The free radical spin trapping agent phenylbutylnitrone reduces fetal brain DNA oxidation and postnatal cognitive deficits caused by in utero exposure to a non-structurally teratogenic dose of ethanol: A role for oxidative stress

Reactive oxygen species (ROS), although implicated in morphological birth defects caused by ethanol (EtOH) during pregnancy, have not been directly linked to its behavioral deficits. To determine this, a pathogenic oxidative DNA lesion was measured in fetal brain, and a passive avoidance learning test was assessed postnatally in the progeny of CD-1 mice treated once on gestational day 17 with 4 g/kg EtOH or its saline vehicle, with or without pretreatment with the free radical spin trapping agent α-phenyl-N-tert-butylnitrone (PBN; 40 mg/kg). EtOH-exposed CD-1 progeny, unlike C57BL/6 progeny, had no morphological birth defects, but exhibited a learning deficit at 12 weeks of age (p<0.001), which continued to 16 weeks in males (p<0.01). Peak blood EtOH concentrations were 2.5-fold higher in C57BL/6 mice compared to CD-1 mice given the same dose. PBN pretreatment of CD-1 dams blocked both EtOH-initiated DNA oxidation in fetal brain (p<0.05) and postnatal learning deficits (p<0.01), providing the first direct evidence for ROS in the mechanism of EtOH-initiated neurodevelopmental deficits.     

Methamphetamine-enhanced embryonic oxidative DNA damage and neurodevelopmental deficits

Methamphetamine (METH) causes dopaminergic nerve terminal degeneration and functional deficits in adult mice, but its neurodevelopmental effects are unclear. We investigated METH-initiated oxidative DNA damage in brain during the embryonic and fetal periods, and the postnatal histological and functional consequences. Pregnant CD-1 mice were treated with a single dose of METH (20 or 40 mg/kg ip) or its saline vehicle on Gestational Day 14 or 17. METH enhanced conceptal DNA oxidation, determined by 8-oxoguanine formation, in brain and liver by at least 2-fold at 1 h (P < 0.05), and more so in some fetal brains at 4 h. After birth, motor coordination on the rotarod apparatus in the METH-exposed offspring was impaired for at least 12 weeks (P < 0.05). Unlike in adults, this postnatal functional deficit in offspring exposed in utero to METH was not associated with degeneration of striatal dopaminergic nerve terminals at 12 weeks of age determined by tyrosine hydroxylase staining, suggesting a novel pathological mechanism in utero. This is the first evidence of oxidative DNA damage in embryonic and fetal brain caused by amphetamines, leading to long-term postnatal neurodevelopmental deficits via a mechanism different from that underlying the neurodegeneration observed in METH-exposed adults.     

Protective mechanisms of Pycnogenol® in ethanol‐insulted cerebellar granule cells

Pycnogenol® (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), inhibits apoptosis and necrosis of developing neurons exposed acutely to ethanol (EtOH). The present study shows that the protective mechanisms of PYC in EtOH‐exposed postnatal day 9 cerebellar granule cells (P9 CGCs) include (1) reduction of reactive oxygen species (ROS) production; (2) counteraction of suppressed copper/zinc superoxide dismutase (Cu/Zn SOD) and glutathione peroxidase/reductase (GSH‐Px/GSSG‐R) system activities; (3) upregulation of Cu/Zn SOD protein expression; (4) mitigation of the EtOH‐mediated exacerbation of catalase (CAT) activity; and, (5) specific binding and inhibition of active caspase‐3. These results indicate that the mechanisms by which PYC antagonizes EtOH‐induced oxidative stress include oxidant scavenging and modulation of endogenous, cellular proteins. Using findings from the present and previous studies, a model delineating the mechanisms of EtOH effects on the system of antioxidant enzymes in developing CGCs is presented. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

The brain‐derived neurotrophic factor VAL68MET polymorphism modulates how developmental ethanol exposure impacts the hippocampus

Prenatal exposure to alcohol causes a wide range of deficits known as fetal alcohol spectrum disorders (FASDs). Many factors determine vulnerability to developmental alcohol exposure including timing and pattern of exposure, nutrition and genetics. Here, we characterized how a prevalent single nucleotide polymorphism in the human brain‐derived neurotrophic factor (BDNF) gene (val66met) modulates FASDs severity. This polymorphism disrupts BDNF's intracellular trafficking and activity‐dependent secretion, and has been linked to increased incidence of neuropsychiatric disorders such as depression and anxiety. We hypothesized that developmental ethanol (EtOH) exposure more severely affects mice carrying this polymorphism. We used transgenic mice homozygous for either valine (BDNF^val/val) or methionine (BDNF^met/met) in residue 68, equivalent to residue 66 in humans. To model EtOH exposure during the second and third trimesters of human pregnancy, we exposed mice to EtOH in vapor chambers during gestational days 12 to 19 and postnatal days 2 to 9. We found that EtOH exposure reduces cell layer volume in the dentate gyrus and the CA1 hippocampal regions of BDNF^met/met but not BDNF^val/val mice during the juvenile period (postnatal day 15). During adulthood, EtOH exposure reduced anxiety‐like behavior and disrupted trace fear conditioning in BDNF^met/met mice, with most effects observed in males. EtOH exposure reduced adult neurogenesis only in the ventral hippocampus of BDNF^val/val male mice. These studies show that the BDNF val66met polymorphism modulates, in a complex manner, the effects of developmental EtOH exposure, and identify a novel genetic risk factor that may regulate FASDs severity in humans.     

Fetal alcohol spectrum disorders among children in a Brazilian orphanage

Background: The objective was to investigate the frequency of fetal alcohol spectrum disorders (FASD) and ophthalmologic anomalies in orphanage children in Brazil. Methods: A prospective study was performed on 94 children living in an orphanage in Brazil. The children were examined by a multidisciplinary team consisting of specialists in pediatrics, neurology, psychology, neuropsychiatry, and ophthalmology. Results: The main reasons for living in the orphanage, in 61% of the children, were negligence, child abuse, and abandonment. Of all the children studied, 50% had mothers with known alcohol abuse and 47% had one or more diagnoses of neurodevelopmental/behavioral and/or cognitive deficits. General developmental delay was found in 18%, intellectual disability in 3%, cognitive impairment in 27%, attention‐deficit/hyperactivity disorder in 14%, and autism in 3%. Altogether 17% had FASD, comprising three children with fetal alcohol syndrome (FAS), six with partial FAS, and seven with alcohol‐related neurodevelopmental disorder. 16% had ophthalmological findings such as poor vision, strabismus, and dysmorphology of the optic nerves. Twenty‐eight children (30%) were adopted from the orphanage; of these, six had FASD (two FAS, three partial FAS, one alcohol‐related neurodevelopmental disorder), five had attention‐deficit/hyperactivity disorder, and eight had developmental delay. Conclusion: Nearly half of the children living in the orphanage had neurodevelopmental disorders and a considerable number showed signs of damage from prenatal alcohol exposure. A broader look at the problem of FASD in Brazil and other South American countries is desirable to document the burden of disease and provide data for targeting prevention efforts. Birth Defects Research (Part A) 103:178–185, 2015. © 2014 Wiley Periodicals, Inc.     

Mechanisms involved in the neurotoxic and cognitive effects of developmental methamphetamine exposure

Methamphetamine exposure in utero leads to a variety of higher‐order cognitive deficits, such as decreased attention and working, and spatial memory impairments in exposed children (Piper et al., 2011; Roussotte et al., 2011; Kiblawi et al., 2011). As with other teratogens, the timing of methamphetamine exposure greatly determines its effects on both neuroanatomical and behavioral outcomes. Methamphetamine exposure in rodents during the third trimester human equivalent period of brain development results in distinct and long‐lasting route‐based and spatial navigation deficits (Williams et al., 2003; Vorhees et al., 2005, 2008, 2009;). Here, we examine the impact of neonatal methamphetamine‐induced neurotoxicity on behavioral outcomes, neurotransmission, receptor changes, plasticity proteins, and DNA damage. Birth Defects Research (Part C) 108:131–141, 2016. © 2016 Wiley Periodicals, Inc.     

Protective mechanisms of pycnogenol in ethanol-insulted cerebellar granule cells

Pycnogenol (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), inhibits apoptosis and necrosis of developing neurons exposed acutely to ethanol (EtOH). The present study shows that the protective mechanisms of PYC in EtOH-exposed postnatal day 9 cerebellar granule cells (P9 CGCs) include (1) reduction of reactive oxygen species (ROS) production; (2) counteraction of suppressed copper/zinc superoxide dismutase (Cu/Zn SOD) and glutathione peroxidase/reductase (GSH-Px/GSSG-R) system activities; (3) upregulation of Cu/Zn SOD protein expression; (4) mitigation of the EtOH-mediated exacerbation of catalase (CAT) activity; and, (5) specific binding and inhibition of active caspase-3. These results indicate that the mechanisms by which PYC antagonizes EtOH-induced oxidative stress include oxidant scavenging and modulation of endogenous, cellular proteins. Using findings from the present and previous studies, a model delineating the mechanisms of EtOH effects on the system of antioxidant enzymes in developing CGCs is presented.     

Melatonin attenuates methamphetamine-induced reduction of tyrosine hydroxylase, synaptophysin and growth-associated protein-43 levels in the neonatal rat brain

Methamphetamine (METH) is a most commonly abused drug which damages nerve terminals by causing formation of reactive oxygen species (ROS), apoptosis, and finally neuronal damage. Fetal exposure to neurotoxic METH causes significant behavioral effects. The developing fetus is substantially deficient in most antioxidative enzymes, and may therefore be at high risk from both endogenous and drug-enhanced oxidative stress. Little is known about the effects of METH on vesicular proteins such as synaptophysin and growth-associated protein 43 (GAP-43) in the immature brain. The present study attempted to investigate the effects of METH-induced neurotoxicity in the dopaminergic system of the neonatal rat brain. Neonatal rats were subcutaneously exposed to 5–10 mg/kg METH daily from postnatal day 4–10 for 7 consecutive days. The results showed that tyrosine hydroxylase enzyme levels were significantly decreased in the dorsal striatum, prefrontal cortex, nucleus accumbens and substantia nigra, synaptophysin levels decreased in the striatum and prefrontal cortex and growth-associated protein-43 (GAP-43) levels significantly decreased in the nucleus accumbens of neonatal rats. Pretreatment with 2 mg/kg melatonin 30 min prior to METH administration prevented METH-induced reduction in tyrosine hydroxylase, synaptophysin and growth-associated protein-43 protein levels in different brain regions. These results suggest that melatonin provides a protective effect against METH-induced nerve terminal degeneration in the immature rat brain probably via its antioxidant properties.     

Dual control of ROS1‐mediated active DNA demethylation by DNA damage‐binding protein 2 (DDB2)

By controlling gene expression, DNA methylation contributes to key regulatory processes during plant development. Genomic methylation patterns are dynamic and must be properly maintained and/or re‐established upon DNA replication and active removal, and therefore require sophisticated control mechanisms. Here we identify direct interplay between the DNA repair factor DNA damage‐binding protein 2 (DDB2) and the ROS1‐mediated active DNA demethylation pathway in Arabidopsis thaliana. We show that DDB2 forms a complex with ROS1 and AGO4 and that they act at the ROS1 locus to modulate levels of DNA methylation and therefore ROS1 expression. We found that DDB2 represses enzymatic activity of ROS1. DNA demethylation intermediates generated by ROS1 are processed by the DNA 3′‐phosphatase ZDP and the apurinic/apyrimidinic endonuclease APE1L, and we also show that DDB2 interacts with both enzymes and stimulates their activities. Taken together, our results indicate that DDB2 acts as a critical regulator of ROS1‐mediated active DNA demethylation.     

Overexpression of human NOX1 complex induces genome instability in mammalian cells

The production of reactive oxygen species (ROS) in mammalian cells is tightly regulated because of their potential to damage macromolecules, including DNA. To investigate possible links between high ROS levels, oxidative DNA damage, and genomic instability in mammalian cells, we established a novel model of chronic oxidative stress by coexpressing the NADPH oxidase human (h) NOX1 gene together with its cofactors NOXO1 and NOXA1. Transfectants of mismatch repair (MMR)-proficient HeLa cells or MMR-defective Msh2(-/-) mouse embryo fibroblasts overexpressing the hNOX1 complex displayed increased intracellular ROS levels. In one HeLa clone in which ROS were particularly elevated, reactive nitrogen species were also increased and nitrated proteins were identified with an anti-3-nitrotyrosine antibody. Overexpression of the hNOX1 complex increased the steady-state levels of DNA 8-oxo-7,8-dihydroguanine and caused a threefold increase in the HPRT mutation rate in HeLa cells. In contrast, additional oxidatively generated damage did not affect the constitutive mutator phenotype of the Msh2(-/-) fibroblasts. Because no significant changes in the expression of several DNA repair enzymes for oxidative DNA damage were identified, we suggest that chronic oxidative stress can saturate the cell's DNA repair capacity and cause significant genomic instability.     

Unwinding protein complexes in ALTernative telomere maintenance

Telomeres are composed of specialized chromatin that includes DNA repair/recombination proteins, telomere DNA‐binding proteins and a number of three dimensional nucleic acid structures including G‐quartets and D‐loops. A number of studies suggest that the BLM and WRN recQ‐like helicases play important roles in recombination‐mediated mechanisms of telomere elongation or A lternative L engthening of T elomeres (ALT), processes that maintain/elongate telomeres in the absence of telomerase. BLM and WRN localize within ALT‐associated nuclear bodies in telomerase‐negative immortalized cell lines and interact with the telomere‐specific proteins POT1, TRF1 and TRF2. Helicase activity is modulated by these interactions. BLM functions in DNA double‐strand break repair processes such as non‐homologous end joining, homologous recombination‐mediated repair, resolution of stalled replication forks and synthesis‐dependent strand annealing, although its precise functions at the telomeres are speculative. WRN also functions in DNA replication, recombination and repair, and in addition to its helicase domain, includes an exonuclease domain not found in other recQ‐like helicases. The biochemical properties of BLM and WRN are, therefore, important in biological processes other than DNA replication, recombination and repair. In this review, we discuss some previous and recent findings of human rec‐Q‐like helicases and their role in telomere elongation during ALT processes. J. Cell. Biochem. 109: 7–15, 2010. © 2009 Wiley‐Liss, Inc.     

The effects of prenatal and postnatal environmental interaction: Prenatal environmental adaptation hypothesis

Adverse antenatal maternal environments during pregnancy influence fetal development that consequently increases risks of mental health problems including psychiatric disorders in offspring. Therefore, behavioral and brain alterations caused by adverse prenatal environmental conditions are generally considered as deficits. In this article, we propose a novel hypothesis, along with summarizing a body of literatures supporting it, that fetal neurodevelopmental alterations, particularly synaptic network changes occurring in the prefrontal cortex, associated with adverse prenatal environmental conditions may be adaptation to cope with expected severe postnatal environments, and therefore, psychiatric disorders may be able to be understood as adaptive strategies against severe environmental conditions through evolution. It is hoped that the hypothesis presented in this article stimulates and opens a new venue on research toward understanding of biological mechanisms and therapeutic treatments of psychiatric disorders.     

Involvement of multiple molecular events in methamphetamine‐induced neurodegeneration: evidence from cDNA array analysis

Methamphetamine (METH) is a neurotoxic drug of abuse that can cause terminal degeneration. Our laboratory recently showed that METH can also cause widespread apoptosis in the rodent brain. Current concepts of the molecular neurotoxicity of this illicit substance have earlier suggested the participation of reactive oxygen species, inflammatory processes and immediate early genes. Recent cDNA studies in our laboratory have hinted to the possibility that METH‐induced neurodegeneration might also include the participation of cell death might also include the participation of cell death genes such as BAX and BCL‐2. Activation of multiple caspases appears to also occur during METH‐induced neurodegeneration. Furthermore, DNA repair pathways seem to also be involved in attempts to protect against METH‐induced DNA damage. These results will be discussed in terms of the possible involvement of multiple transduction mechanisms in the appearance of METH neurotoxicity.     

Increased aggression and lack of maternal behavior in Dio3‐deficient mice are associated with abnormalities in oxytocin and vasopressin systems

Thyroid hormones regulate many aspects of brain development and function, and alterations in the levels of thyroid hormone action lead to abnormal anxiety‐ and depression‐like behaviors. A complement of factors in the brain function independently of circulating levels of hormone to strictly controlled local thyroid hormone signaling. A critical factor is the type 3 deiodinase (DIO3), which is located in neurons and protects the brain from excessive thyroid hormone. Here, we examined whether a local increase in brain thyroid hormone action secondary to DIO3 deficiency is of consequence for social behaviors. Although we did not observe alterations in sociability, Dio3?/? mice of both sexes exhibited a significant increase in aggression‐related behaviors and mild deficits in olfactory function. In addition, 85% of Dio3?/? dams manifested no pup‐retrieval behavior and increased aggression toward the newborns. The abnormal social behaviors of Dio3?/? mice were associated with sexually dimorphic alterations in the physiology of oxytocin (OXT) and arginine vasopressin (AVP), 2 neuropeptides with important roles in determining social interactions. These alterations included low adult serum levels of OXT and AVP, and an abnormal expression of Oxt, Avp and their receptors in the neonatal and adult hypothalamus. Our results demonstrate that DIO3 is essential for normal aggression and maternal behaviors, and indicate that abnormal local regulation of thyroid hormone action in the brain may contribute to the social deficits associated with neurodevelopmental disorders.     

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

Base excision repair （BER） is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged （oxidized or aikylated） or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species （ROS）. BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease （APE） to generate 3＇ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA iigase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APEI, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3＇ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and iigases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organeile targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.     

A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny

Genes that promote DNA methylation and demethylation in plants have been characterized mainly in Arabidopsis. Arabidopsis DNA demethylation is mediated by bi-functional DNA enzymes with glycosylase activity that removes 5-methylcytosine and lyase activity that nicks double-stranded DNA at an abasic site. Homologous recombination-promoted knock-in targeting of the ROS1a gene, the longest of six putative DNA demethylase genes in the rice genome, by fusing its endogenous promoter to the GUS reporter gene, led to reproducibly disrupted ROS1a in primary (T(0)) transgenic plants in the heterozygous condition. These T(0) plants exhibited no overt morphological phenotypes during the vegetative phase, and GUS staining showed ROS1a expression in pollen, unfertilized ovules and meristematic cells. Interestingly, neither the maternal nor paternal knock-in null allele, ros1a-GUS1, was virtually detected in the progeny; such an intransmittable null mutation is difficult to isolate by conventional mutagenesis techniques that are usually used to identify and isolate mutants in the progeny population. Even in the presence of the wild-type paternal ROS1a allele, the maternal ros1a-GUS1 allele caused failure of early-stage endosperm development, resulting in incomplete embryo development, with embryogenesis producing irregular but viable embryos that failed to complete seed dormancy, implying non-equivalent maternal and paternal contribution of ROS1a in endosperm development. The paternal ros1a-GUS1 allele was not transmitted to progeny, presumably because of a male gametophytic defect(s) prior to fertilization. Thus, ROS1a is indispensable in both male and female gametophytes, and DNA demethylation must plays important roles in both gametophytes.     

Proteomic analyses of ethanol tolerance in Lactobacillus buchneri NRRL B‐30929

The Lactobacillus buchneri NRRL B‐30929 strain, isolated from a fuel ethanol (EtOH) production facility, exhibits high tolerance to environmental EtOH concentrations. This study aimed to identify proteins produced by B‐30929 in response to environmental EtOH. Cellular proteins expressed by B‐30929 growing in media with 10 versus 0% EtOH were compared by 2DE, followed by in‐gel digestion and MALDI‐MS analyses. Twenty EtOH responsive proteins were identified. These include a proline‐specific peptidase (Lbuc_1852); a membrane protein (Lbuc_0921), two general stress‐related proteins including a 10 kDa chaperonin (GroESL Lbuc_1359) and a 29 kDa member of the HK 97 family (Lbuc_1523); metabolic enzymes involving redox potential balances (Lbuc_2051 and Lbuc_0522) and carbohydrate fermentation (Lbuc_1319 and Lbuc_2157); nitrogen, amino acid, and fatty acid metabolism proteins (Lbuc_1994, Lbuc_0446, Lbuc_0858, Lbuc_0707, and Lbuc_0787). These changes suggested B‐30929 cells respond to EtOH by degradation of available proteins and fatty acids and increased production of specific enzymes and molecular chaperons. These results can be used to guide genetic modifications to increase EtOH tolerance in industrial biocatalysts. The data have been deposited to World‐2DPAGE ( http://world‐2dpage.expasy.org/repository/0068/ ; username liu, password 1h8d6Mg1).