A new blood based epigenetic age predictor for adolescents and young adults.

Children have special rights for protection compared to adults in our society. However, more than 1/4 of children globally have no documentation of their date of birth. Hence, there is a pressing need to develop biological methods for chronological age prediction, robust to differences in genetics, psychosocial events and physical living conditions. At present, DNA methylation is the most promising biological biomarker applied for age assessment. The human genome contains around 28 million DNA methylation sites, many of which change with age. Several epigenetic clocks accurately predict chronological age using methylation levels at age associated GpG-sites. However, variation in DNA methylation increases with age, and there is no epigenetic clock specifically designed for adolescents and young adults. Here we present a novel age Predictor for Adolescents and Young Adults (PAYA), using 267 CpG methylation sites to assess the chronological age of adolescents and young adults. We compared different preprocessing approaches and investigated the effect on prediction performance of the epigenetic clock. We evaluated performance using an independent validation data set consisting of 18-year-old individuals, where we obtained a median absolute deviation of just below 0.7 years. This tool may be helpful in age assessment of adolescents and young adults. However, there is a need to investigate the robustness of the age predictor across geographical and disease populations as well as environmental effects.

NEIL1 and NEIL2 DNA glycosylases modulate anxiety and learning in a cooperative manner in mice.

Oxidative DNA damage in the brain has been implicated in neurodegeneration and cognitive decline. DNA glycosylases initiate base excision repair (BER), the main pathway for oxidative DNA base lesion repair. NEIL1 and NEIL3 DNA glycosylases affect cognition in mice, while the role of NEIL2 remains unclear. Here, we investigate the impact of NEIL2 and its potential overlap with NEIL1 on behavior in knockout mouse models. Neil1-/-Neil2-/- mice display hyperactivity, reduced anxiety and improved learning. Hippocampal oxidative DNA base lesion levels are comparable between genotypes and no mutator phenotype is found. Thus, impaired canonical repair is not likely to explain the altered behavior. Electrophysiology suggests reduced axonal activation in the hippocampal CA1 region in Neil1-/-Neil2-/- mice and lack of NEIL1 and NEIL2 causes dysregulation of genes in CA1 relevant for synaptic function. We postulate a cooperative function of NEIL1 and NEIL2 in genome regulation, beyond canonical BER, modulating behavior in mice.

Advancing estimation of chronological age by utilizing available evidence based on two radiographical methods.

This paper describes a strategy for estimating chronological age of individuals based on age indicators of X-ray of the hand and the third molar tooth. The great majority of studies in the field provide group-wise data of different formats, which makes them difficult to compare and utilize in a model. In this paper, we have provided a framework to utilize different types of data formats to build a common model for estimating chronological age. We used transition analysis to describe the relationship between the age indicators and chronological age. Further, likelihood ratio weight of evidence and posterior distribution of chronological age were used to model the distribution of chronological age given the observed age indicators. Being able to utilize such a large amount of data, with different data formats, from different studies, as presented in this paper improves previous age estimation methods.

BioAlder: a tool for assessing chronological age based on two radiological methods.

We have created the tool BioAlder as an age prediction model based on the systems Greulich and Pyle (hand) and the Demirjian's grading of the third molar tooth. The model compiles information from studies representing a total of 17,151 individuals from several parts of the world. The model offers a solution where issues as group-wise data format and age mimicry bias are bypassed. The model also provides a solution for combining the two grading systems, hand and tooth, to one combined age prediction result assuming independency. We have tested our model of age prediction and the independency assumption on a separate data set from Lebanon with 254 young individuals. The prediction intervals of BioAlder covered most of the data points; however, we observed some outliers. Our analyses indicate at least a weak dependency between the two methods.

A systematic review of the agreement between chronological age and skeletal age based on the Greulich and Pyle atlas.

OBJECTIVES: This systematic review examines the agreement between assessed skeletal age by the Greulich and Pyle atlas (GP skeletal age) and chronological age. METHODS: We searched electronic databases until January 2017 for studies reporting GP skeletal age and confirmed chronological age in healthy individuals aged 10-25 years. Results are presented as forest plots and meta-analyses (random-effects models). RESULTS: In separate meta-analyses for each age group and sex (14-18 years for girls, 14-19 years for boys), the pooled mean differences between GP skeletal age and chronological age varied from -0.52 years to 0.47 years. In individual studies, age group and sex-specific mean differences between GP skeletal age and chronological age rarely exceeded 1 year, but between-study heterogeneities were large in most age groups. Few studies examined mean chronological age and distribution for each GP skeletal age. One study of good methodological quality indicates that 95% prediction intervals for chronological age from given GP skeletal ages are typically around 4 years. CONCLUSIONS: There is still good correlation between GP skeletal age and mean chronological age in modern populations. However, the individual variation of development within a population and heterogeneities between studies are substantial. KEY POINTS: • The GP atlas still corresponds well with mean chronological age in modern populations. • The substantial variation within a population must be considered. • The heterogeneity between studies is relatively large and of unknown origin.

Age assessment by Demirjian's development stages of the third molar: a systematic review.

OBJECTIVES: Radiographic evaluation of the wisdom teeth (third molar) formation is a widely used age assessment method for adolescents and young adults. This systematic review examines evidence on the agreement between Demirjian's development stages of the third molar and chronological age. METHODS: We searched four databases up until May 2016 for studies reporting Demirjian's stages of third molar and confirmed chronological age of healthy individuals aged 10-25 years. Heterogeneity test of the included studies was performed. RESULTS: We included 21 studies from all continents except Australia, all published after 2005. The mean chronological age for Demirjian's stages varied considerably between studies. The results from most studies were affected by age mimicry bias. Only a few of the studies based their results on an unbiased age structure, which we argue as important to provide an adequate description of the method's ability to estimate age. CONCLUSION: Observed study variation in the timing of Demirjian's development stages for third molars has often been interpreted as differences between populations and ethnicities. However, we consider age mimicry to be a dominant bias in these studies. Hence, the scientific evidence is insufficient to conclude whether such differences exist. KEY POINTS: • There is significant heterogeneity between studies evaluating age assessment by Demirjian's third molar development. • Most of the studies were subject to the selection bias age mimicry which can be a source of heterogeneity. • Presence of age mimicry bias makes it impossible to compare and combine results. These biased studies should not be applied as reference studies for age assessment.

No cancer predisposition or increased spontaneous mutation frequencies in NEIL DNA glycosylases-deficient mice.

Base excision repair (BER) is a major pathway for removal of DNA base lesions and maintenance of genomic stability, which is essential in cancer prevention. DNA glycosylases recognize and remove specific lesions in the first step of BER. The existence of a number of these enzymes with overlapping substrate specificities has been thought to be the reason why single knock-out models of individual DNA glycosylases are not cancer prone. In this work we have characterized DNA glycosylases NEIL1 and NEIL2 (Neil1 -/- /Neil2 -/-) double and NEIL1, NEIL2 and NEIL3 (Neil1 -/- /Neil2 -/- /Neil3 -/-) triple knock-out mouse models. Unexpectedly, our results show that these mice are not prone to cancer and have no elevated mutation frequencies under normal physiological conditions. Moreover, telomere length is not affected and there was no accumulation of oxidative DNA damage compared to wild-type mice. These results strengthen the hypothesis that the NEIL enzymes are not simply back-up enzymes for each other but enzymes that have distinct functions beyond canonical repair.

Synergistic Actions of Ogg1 and Mutyh DNA Glycosylases Modulate Anxiety-like Behavior in Mice.

Ogg1 and Mutyh DNA glycosylases cooperate to prevent mutations caused by 8-oxoG, a major premutagenic DNA lesion associated with cognitive decline. We have examined behavior and cognitive function in mice deficient of these glycosylases. Ogg1(-/-)Mutyh(-/-) mice were more active and less anxious, with impaired learning ability. In contrast, Mutyh(-/-) mice showed moderately improved memory. We observed no apparent change in genomic 8-oxoG levels, suggesting that Ogg1 and Mutyh play minor roles in global repair in adult brain. Notably, transcriptome analysis of hippocampus revealed that differentially expressed genes in the mutants belong to pathways known to be involved in anxiety and cognition. Esr1 targets were upregulated, suggesting a role of Ogg1 and Mutyh in repression of Esr1 signaling. Thus, beyond their involvement in DNA repair, Ogg1 and Mutyh regulate hippocampal gene expression related to cognition and behavior, suggesting a role for the glycosylases in regulating adaptive behavior.

Loss of Neil3, the major DNA glycosylase activity for removal of hydantoins in single stranded DNA, reduces cellular proliferation and sensitizes cells to genotoxic stress.

7,8-Dihydro-8-oxoguanine (8-oxoG) is one of the most common oxidative base lesions in normal tissues induced by a variety of endogenous and exogenous agents. Hydantoins are products of 8-oxoG oxidation and as 8-oxoG, they have been shown to be mutagenic lesions. Oxidative DNA damage has been implicated in the etiology of various age-associated pathologies, such as cancer, cardiovascular diseases, arthritis, and several neurodegenerative diseases. The mammalian endonuclease VIII-like 3 (Neil3) is one of the four DNA glycosylases found to recognize and remove hydantoins in the first step of base excision repair (BER) pathway. We have generated mice lacking Neil3 and by using total cell extracts we demonstrate that Neil3 is the main DNA glycosylase that incises hydantoins in single stranded DNA in tissues. Using the neurosphere culture system as a model to study neural stem/progenitor (NSPC) cells we found that lack of Neil3 impaired self renewal but did not affect differentiation capacity. Proliferation was also reduced in mouse embryonic fibroblasts (MEFs) derived from Neil3(-/-) embryos and these cells were sensitive to both the oxidative toxicant paraquat and interstrand cross-link (ICL)-inducing agent cisplatin. Our data support the involvement of Neil3 in removal of replication blocks in proliferating cells.

Hippocampal adult neurogenesis is maintained by Neil3-dependent repair of oxidative DNA lesions in neural progenitor cells.

Accumulation of oxidative DNA damage has been proposed as a potential cause of age-related cognitive decline. The major pathway for removal of oxidative DNA base lesions is base excision repair, which is initiated by DNA glycosylases. In mice, Neil3 is the main DNA glycosylase for repair of hydantoin lesions in single-stranded DNA of neural stem/progenitor cells, promoting neurogenesis. Adult neurogenesis is crucial for maintenance of hippocampus-dependent functions involved in behavior. Herein, behavioral studies reveal learning and memory deficits and reduced anxiety-like behavior in Neil3(-/-) mice. Neural stem/progenitor cells from aged Neil3(-/-) mice show impaired proliferative capacity and reduced DNA repair activity. Furthermore, hippocampal neurons in Neil3(-/-) mice display synaptic irregularities. It appears that Neil3-dependent repair of oxidative DNA damage in neural stem/progenitor cells is required for maintenance of adult neurogenesis to counteract the age-associated deterioration of cognitive performance.

Endonuclease VIII-like 3 (Neil3) DNA glycosylase promotes neurogenesis induced by hypoxia-ischemia.

Neural stem/progenitor cell proliferation and differentiation are required to replace damaged neurons and regain brain function after hypoxic-ischemic events. DNA base lesions accumulating during hypoxic-ischemic stress are removed by DNA glycosylases in the base-excision repair pathway to prevent cytotoxicity and mutagenesis. Expression of the DNA glycosylase endonuclease VIII-like 3 (Neil3) is confined to regenerative subregions in the embryonic and perinatal brains. Here we show profound neuropathology in Neil3-knockout mice characterized by a reduced number of microglia and loss of proliferating neuronal progenitors in the striatum after hypoxia-ischemia. In vitro expansion of Neil3-deficient neural stem/progenitor cells revealed an inability to augment neurogenesis and a reduced capacity to repair for oxidative base lesions in single-stranded DNA. We propose that Neil3 exercises a highly specialized function through accurate molecular repair of DNA in rapidly proliferating cells.

Widespread distribution of DNA glycosylases removing oxidative DNA lesions in human and rodent brains.

High metabolic activity and low levels of antioxidant enzymes make neurons particularly prone to damage by reactive oxygen species. Thus, repair of oxidative DNA damage is essential for normal brain function. Base excision repair is the major pathway for repair of oxidative DNA damage, and is initiated by DNA glycosylases recognizing and removing the damaged base. In mammalian cells at least five different DNA glycosylases with overlapping substrate specificity, NEIL1, NEIL2, NEIL3, OGG1 and NTH1, remove oxidative DNA base lesions. Here we report mRNA expression and distribution of these five DNA glycosylases in human and rodent brains using in situ hybridization and Northern blotting supported by glycosylase activity assays. NEIL1, NEIL2, OGG1 and NTH1 showed widespread expression at all ages. In situ hybridization studies in mouse brain showed that expression of mNeil1 increased with age. In newborn mouse brain, mNeil3 revealed a discrete expression pattern in brain regions known to harbour stem cell populations, i.e., the subventricular zone, the rostral migratory stream, and the hilar region of the hippocampal formation. Expression of mNeil3 decreased with age, and in old mice brains could be detected only in layer V of neocortex. MNth1 was constitutively expressed during lifespan. In Northern blots, mOgg1 expression showed a transient decrease followed by an increase after 8 weeks of age. Assays for faPy DNA glycosylase activity revealed increased activity level with age in all brain regions analyzed. The widespread but differential expression of the DNA glycosylases recognizing oxidative base lesions suggests distinct and age dependent roles of these enzymes in genome maintenance in brain. The distribution of mNeil3 is particularly intriguing and points to a specific role of this enzyme in stem cell differentiation.

Base excision repair activities in organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation.

The capacity for DNA repair is likely to be one of the factors that determine the vulnerability of neurons to ischemic stress and may influence the pathological outcome of stroke. In this report, initiation of base excision repair (BER) was assessed by analysis of enzyme activity and gene expression level of DNA glycosylases and AP-endonucleases in rat organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD) - an in vitro model of stroke. Under basal conditions, AP-endonuclease activity and base removal of ethenoadenine and 8-oxoguanine (8-oxoG) were higher (by approximately 20-35 %) in CA3/fascia dentata (FD) than in CA1. Base removal of uracil did not differ between the two hippocampal regions, while removal of 5-hydroxyuracil (5-OHU) was slightly less efficient in CA3/FD than in CA1. Analyses performed immediately after 30 min of OGD revealed a decreased AP-endonuclease activity (by approximately 20%) in CA1 as well as CA3/FD, and an increased ethenoadenine activity (by approximately 25%) in CA1. Activities for 8-oxoG, 5-OHU and uracil showed no significant changes at this time point. At 8h after OGD, none of the enzyme activities differed from control values. Real-time RT-PCR showed that transcription of DNA glycosylases, including Ogg1, Nth1, Ung, Aag, Neil1 and Neil2 were not changed in response to OGD treatment (t=0 h). The hippocampal expression of Neil2 was low compared with the other DNA glycosylases. These data indicate that CA1 has a lower capacity than CA3/FD for removal of base lesions under basal conditions. The relatively low capacity for BER in basal conditions and the apparent failure to upregulate repair of oxidative damage after OGD might contribute to the high vulnerability of CA1 to ischemic injury.

Human NEIL1 localizes with the centrosomes and condensed chromosomes during mitosis.

The DNA glycosylase hNEIL1 initiates base excision repair (BER) of a number of oxidized purines and pyrimidines in cellular DNA and is one of three mammalian orthologs of the Escherichia coli Nei/Fpg enzymes. Human NEIL1 has been purified and extensively characterized biochemically, however, not much is known about its intracellular distribution. In the present work, we have studied the cellular localization of hNEIL1 using both antibodies raised against the full-length recombinant protein and a stable HeLa cell line expressing hNEIL1 fused N-terminal to EGFP. The results presented reveal an intricate mitotic distribution of hNEIL1. Centrosomal localization of hNEIL1 was observed when mitotic HeLa cells were immunostained with hNEIL1 antibodies. This localization was confirmed when Western blots of isolated centrosomes from stably expressing hNEIL1-EGFP HeLa cells were probed with GFP or hNEIL1 antibodies, even though a fluorescent signal could not be detected in the centrosomes of these cells. Human NEIL1 was also shown to be associated with mitotic condensed chromosomes. Notably, the interaction of hNEIL1 with condensed chromatin was disrupted when cells were fixed with chemical fixatives that are regularly used in immunodetection techniques.

Dynamic relocalization of hOGG1 during the cell cycle is disrupted in cells harbouring the hOGG1-Cys326 polymorphic variant.

Numerous lines of evidence support the role of oxidative stress in different types of cancer. A major DNA lesion, 8-oxo-7,8-dihydroguanine (8-oxoG), is formed by reactive oxygen species in the genome under physiological conditions. 8-OxoG is strongly mutagenic, generating G.C-->T.A transversions, a frequent somatic mutation in cancers. hOGG1 was cloned as a gene encoding a DNA glycosylase that specifically recognizes and removes 8-oxoG from 8-oxoG:C base pairs and suppresses G.C-->T.A transversions. In this study, we investigated the subcellular localization and expression of hOGG1 during the cell cycle. Northern blots showed cell-cycle-dependent mRNA expression of the two major hOGG1 isoforms. By using a cell line constitutively expressing hOGG1 fused to enhanced green fluorescence protein (EGFP), we observed a dynamic relocalization of EGFP-hOGG1 to the nucleoli during the S-phase of the cell cycle, and this localization was shown to be linked to transcription. A C/G change that results in an amino acid substitution from serine to cysteine in codon 326 has been reported as a genetic polymorphism and a risk allele for a variety of cancers. We investigated the cellular localization of the corresponding protein, hOGG1-Cys326, fused to EGFP and observed a dramatic effect on its localization that is explained by a change in the phosphorylation status of hOGG1.

Human DNA glycosylases of the bacterial Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA.

The mild phenotype associated with targeted disruption of the mouse OGG1 and NTH1 genes has been attributed to the existence of back-up activities and/or alternative pathways for the removal of oxidised DNA bases. We have characterised two new genes in human cells that encode DNA glycosylases, homologous to the bacterial Fpg (MutM)/Nei class of enzymes, capable of removing lesions that are substrates for both hOGG1 and hNTH1. One gene, designated HFPG1, showed ubiquitous expression in all tissues examined whereas the second gene, HFPG2, was only expressed at detectable levels in the thymus and testis. Transient transfections of HeLa cells with fusions of the cDNAs to EGFP revealed intracellular sorting to the nucleus with accumulation in the nucleoli for hFPG1, while hFPG2 co-localised with the 30 kDa subunit of RPA. hFPG1 was purified and shown to act on DNA substrates containing 8-oxoguanine, 5-hydroxycytosine and abasic sites. Removal of 8-oxoguanine, but not cleavage at abasic sites, was opposite base-dependent, with 8-oxoG:C being the preferred substrate and negligible activity towards 8-oxoG:A. It thus appears that hFPG1 has properties similar to mammalian OGG1 in preventing mutations arising from misincorporation of A across 8-oxoG and could function as a back-up repair activity for OGG1 in ogg1(-/-) mice.

Additive toxicity of limonene and 50% oxygen and the role of glutathione in detoxification in human lung cells.

Limonene has many commercial applications and has been introduced as an environmentally acceptable solvent replacing halogenated hydrocarbons. Occupational exposure to limonene presumably occurs simultaneously with other chemicals including oxidative agents and may exert a heavy strain on cellular detoxifying capacity resulting in synergistic effects. The present study used oxygen as an example of an ubiquitous oxidative and radical forming agent and investigated the combination effects with limonene on human lung cells. Mechanistic information was gained by comparing the toxicity of limonene with a major oxidation product, limonene 1,2-epoxide, and by the involvement of glutathione in cellular detoxification. At cell culture conditions most similar to the in vivo situation oxygen did not increase the toxicity of limonene beyond an additive effect. The results further indicated that limonene 1,2-epoxide was not the active compound in limonene toxicity. Experimental evidence suggests that detoxification of limonene in human lung cells primarily occurs by mechanisms not involving the glutathione system and point to possible long-term effects of limonene exposure. The present knowledge indicates clearly that the mechanism of action of limonene on biological systems and particularly in combination with oxidative compounds still remains to be elucidated. In light of the frequent exposure of humans to such combinations further investigations into this issue are highly recommended.