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DNA methylation plays a pivotal role in cancer. The ubiquitous contaminant perfluorooctanesulfonic acid (PFOS) has been epidemiologically associated with breast cancer, and can induce proliferation and malignant transformation of normal human breast epithelial cells (MCF-10A), but the information about its effect on DNA methylation is sparse. The aim of this study was to characterize the whole-genome methylome effects of PFOS in our breast cell model and compare the findings with previously demonstrated DNA methylation alterations in breast tumor tissues. The DNA methylation profile was assessed at single CpG resolution in MCF-10A cells treated with 1 μM PFOS for 72 h by using Enzymatic Methyl sequencing (EM-seq). We found 12,591 differentially methylated CpG-sites and 13,360 differentially methylated 100 bp tiles in the PFOS exposed breast cells. These differentially methylated regions (DMRs) overlapped with 2406 genes of which 494 were long non-coding RNA and 1841 protein coding genes. We identified 339 affected genes that have been shown to display altered DNA methylation in breast cancer tissue and several other genes related to cancer development. This includes hypermethylation of GACAT3, DELEC1, CASC2, LCIIAR, MUC16, SYNE1 and hypomethylation of TTN and KMT2C. DMRs were also found in estrogen receptor genes (ESR1, ESR2, ESRRG, ESRRB, GREB1) and estrogen responsive genes (GPER1, EEIG1, RERG). The gene ontology analysis revealed pathways related to cancer phenotypes such as cell adhesion and growth. These findings improve the understanding of PFOS's potential role in breast cancer and illustrate the value of whole-genome methylome analysis in uncovering mechanisms of chemical effects, identifying biomarker candidates, and strengthening epidemiological associations, potentially impacting risk assessment., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The unsustainable use of manmade chemicals poses significant threats to biodiversity and human health. Emerging evidence highlights the potential of certain chemicals to cause transgenerational impacts on metabolic health. Here, we investigate male transmitted epigenetic transgenerational effects of the anti-androgenic herbicide linuron in the pancreas of Xenopus tropicalis frogs, and their association with metabolic phenotypes. Reduced representation bisulfite sequencing (RRBS) was used to assess genome-wide DNA methylation patterns in the pancreas of adult male F2 generation ancestrally exposed to environmentally relevant linuron levels (44 ± 4.7 μg/L). We identified 1117 differentially methylated regions (DMRs) distributed across the X. tropicalis genome, revealing potential regulatory mechanisms underlying metabolic disturbances. DMRs were identified in genes crucial for pancreatic function, including calcium signalling (clstn2, cacna1d and cadps2), genes associated with type 2 diabetes (tcf7l2 and adcy5) and a biomarker for pancreatic ductal adenocarcinoma (plec). Correlation analysis revealed associations between DNA methylation levels in these genes and metabolic phenotypes, indicating epigenetic regulation of glucose metabolism. Moreover, differential methylation in genes related to histone modifications suggests alterations in the epigenetic machinery. These findings underscore the long-term consequences of environmental contamination on pancreatic function and raise concerns about the health risks associated with transgenerational effects of pesticides., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Cecilia Berg is now employed at The Swedish Medical Products Agency, and Sofie Svanholm is now employed at The Swedish Chemicals Agency, but The Swedish Medical Products Agency or The Swedish Chemicals Agency were not involved in any part of this study. All work was carried out when employed at Uppsala University. The views expressed in this study are that of the authors alone and do not reflect those of The Swedish Medical Products Agency or The Swedish Chemicals Agency., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The herbicide linuron can cause endocrine disrupting effects in Xenopus tropicalis frogs, including offspring that were never exposed to the contaminant. The mechanisms by which these effects are transmitted across generations need to be further investigated. Here, we examined transgenerational alterations of brain and testis DNA methylation profiles paternally inherited from grandfathers developmentally exposed to an environmentally relevant concentration of linuron. Reduced representation bisulfite sequencing (RRBS) revealed numerous differentially methylated regions (DMRs) in brain (3060 DMRs) and testis (2551 DMRs) of the adult male F2 generation. Key genes in the brain involved in somatotropic (igfbp4) and thyrotropic signaling (dio1 and tg) were differentially methylated and correlated with phenotypical alterations in body size, weight, hind limb length and plasma glucose levels, indicating that these methylation changes could be potential mediators of the transgenerational effects of linuron. Testis DMRs were found in genes essential for spermatogenesis, meiosis and germ cell development (piwil1, spo11 and tdrd9) and their methylation levels were correlated with the number of germ cells nests per seminiferous tubule, an endpoint of disrupted spermatogenesis. DMRs were also identified in several genes central for the machinery that regulates the epigenetic landscape including DNA methylation (dnmt3a and mbd2) and histone acetylation (hdac8, ep300, elp3, kat5 and kat14), which may at least partly drive the linuron-induced transgenerational effects. The results from this genome-wide DNA methylation profiling contribute to better understanding of potential transgenerational epigenetic inheritance mechanisms in amphibians., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. All work was carried out when employed at Uppsala University. Sofie Svanholm is now employed at The Swedish Chemicals Agency but The Swedish Chemicals Agency was not involved in any part of this study. The views expressed in this study are that of the author alone and do not reflect those of the Swedish Chemicals Agency., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Early life exposure to endocrine disrupting chemicals (EDCs) has been suggested to adversely affect reproductive health in humans and wildlife. Here, we characterize endocrine and adverse effects on the reproductive system after juvenile exposure to propiconazole (PROP) or imazalil (IMZ), two common azole fungicides with complex endocrine modes of action. Using the frog Xenopus tropicalis, two short-term (2-weeks) studies were conducted. I: Juveniles (2 weeks post metamorphosis (PM)) were exposed to 0, 17 or 178 µg PROP/L. II: Juveniles (6 weeks PM) were exposed to 0, 1, 12 or 154 µg IMZ/L. Histological analysis of the gonads revealed an increase in the number of dark spermatogonial stem cells (SSCs)/testis area, and in the ratio secondary spermatogonia: dark SSCs were increased in all IMZ groups compared to control. Key genes in gametogenesis, retinoic acid and sex steroid pathways were also analysed in the gonads. Testicular levels of 3β-hsd, ddx4 were increased and cyp19 and id4 levels were decreased in the IMZ groups. In PROP exposed males, increased testicular aldh1a2 levels were detected, but no histological effects observed. Although no effects on ovarian histology were detected, ovarian levels of esr1, rsbn1 were increased in PROP groups, and esr1 levels were decreased in IMZ groups. In conclusion, juvenile azole exposure disrupted testicular expression of key genes in retinoic acid (PROP) and sex steroid pathways and in gametogenesis (IMZ). Our results further show that exposure to environmental concentrations of IMZ disrupted spermatogenesis in the juvenile testis, which is a cause for concern as it may lead to impaired fertility. Testicular levels of id4, ddx4 and the id4:ddx4 ratio were associated with the number of dark SSCs and secondary spermatogonia suggesting that they may serve as a molecular markers for disrupted spermatogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Di-n-butyl phthalate (DBP) is a ubiquitous environmental contaminant linked with various adverse health effects, including immune system dysfunction. Gut microbial dysbiosis can contribute to a wide range of pathogenesis, particularly immune disease. Here, we investigated the impact of DBP on the gut microbiome and examined correlations with immune system changes after five weeks oral exposure (10 or 100 mg/kg/day) in adult male mice. The fecal microbiome composition was characterized using 16S rRNA sequencing. DBP-treated mice displayed a significantly distinct microbial community composition, indicated by Bray-Curtis distance. Numerous amplicon sequence variants (ASVs) at the genus level were altered. Compared to the vehicle control group, the 10 mg/kg/day DBP group had 63 more abundant and 65 less abundant ASVs, while 60 ASVs were increased and 76 ASVs were decreased in the 100 mg/kg/day DBP group. Both DBP treatment groups showed higher abundances of ASVs assigned to Desulfovibrio (Proteobacteria phylum) and Enterorhabdus genera, while ASVs belonging to Parabacteroides, Lachnospiraceae UCG-006 and Lachnoclostridium were less common compared to the control group. Interestingly, an ASV belonging to Rumniniclostridium 6, which was less abundant in DBP-treated mice, demonstrated a negative correlation with the increased number of non-classical monocytes observed in the blood of DBP-treated animals. In addition, an ASV from Lachnospiraceae UCG-001, which was more abundant in the DBP-treated animals, showed a positive correlation with the non-classical monocyte increase. This study shows that DBP exposure greatly modifies the gut bacterial microbiome and indicates a potential contribution of microbial dysbiosis to DBP-induced immune system impairment, illustrating the importance of investigating how interactions between exposome components can affect health., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Increased exposure to manmade chemicals may be linked to an increase in immune-related diseases in humans and immune system dysfunction in wildlife. Phthalates are a group of endocrine-disrupting chemicals (EDCs) suspected to influence the immune system. The aim of this study was to characterize the persistent effects on leukocytes in the blood and spleen, as well as plasma cytokine and growth factor levels, one week after the end of five weeks of oral treatment with dibutyl phthalate (DBP; 10 or 100 mg/kg/d) in adult male mice. Flow cytometry analysis of the blood revealed that DBP exposure decreased the total leukocyte count, classical monocyte and T helper (Th) populations, whereas it increased the non-classical monocyte population compared to the vehicle control (corn oil). Immunofluorescence analysis of the spleen showed increased CD11b + Ly6G + (marker of polymorphonuclear myeloid-derived suppressor cells; PMN-MDSCs), and CD43 + staining (marker of non-classical monocytes), whereas CD3 + (marker of total T cells) and CD4 + (marker of Th cells) staining decreased. To investigate the mechanisms of action, levels of plasma cytokines and chemokines were measured using multiplexed immunoassays and other key factors were analyzed using western blotting. The observed increase in M-CSF levels and the activation of STAT3 may promote PMN-MDSC expansion and activity. Increased ARG1, NOX2 (gp91phox), and protein nitrotyrosine levels, as well as GCN2 and phosphor-eIRFα, suggest that oxidative stress and lymphocyte arrest drive the lymphocyte suppression caused by PMN-MDSCs. The plasma levels of IL-21 (promotes the differentiation of Th cells) and MCP-1 (regulates migration and infiltration of monocytes/macrophages) also decreased. These findings show that adult DBP exposure can cause persistent immunosuppressive effects, which may increase susceptibility to infections, cancers, and immune diseases, and decrease vaccine efficacy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

It is important to identify the factors that influence the prevalence of disinhibitory behaviors, as tobacco and alcohol use in adolescence is a strong predictor of continued use and substance abuse into adulthood. Organochlorine pesticides (OCPs) are persistent organic pollutants that pose a potential risk to the developing fetus and offspring long-term health. We examined associations between prenatal exposure OCPs and their metabolites (i.e., p,p'-DDT, p,p'-DDE, o,p'-DDT, oxychlordane, and hexachlorobenzene (HCB)), both as a mixture and single compounds, and alcohol consumption and smoking at adolescence in a sample (n = 554) from the Child Health and Development Studies prospective birth cohort. Bayesian Kernel Machine Regression demonstrated a trend of higher risk of alcohol use and smoking with higher quartile mixture levels. Single-component analysis showed increased odds of smoking and drinking with increases in lipid-adjusted p,p'-DDE serum levels (aOR = 2.06, 95% CI 0.99-4.31, p = 0.05, per natural log unit increase). We found significant effect modification in these associations by sex with higher p,p'-DDT serum levels (aOR = 0.26, 95% CI 0.09-0.076, p = 0.01, per natural log unit increase) was associated with lower odds of smoking and drinking in female adolescents, while higher p,p'-DDE serum levels (aOR = 2.98, 95% CI 1.04-8.51, p = 0.04, per natural log unit increase) was associated with higher odds of the outcomes. Results of the mutually adjusted model were not significant for male adolescents. Further research to understand reasons for these sex-differences are warranted., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Environmental contaminants including long-chain per- and polyfluoroalkyl substances (PFAS) have been linked to cancer, which is a central cause of mortality in humans and many wildlife species. Today shorter-chain PFAS are extensively used as replacement compounds and commonly found in the environment. Mechanistic studies are important for a better understanding of their toxicological potential and possible role in cancer etiology. Here, we treated normal human breast epithelial cells (MCF-10A) with 500 pM to 500 μM of perfluorohexane sulfonate (PFHxS), undecafluorohexanoic acid (PFHxA), hexafluoropropylene oxide-dimer acid (GenX), perfluoro 3,6 dioxaoctanoic acid (PFO2OA), heptafluorobutyric acid (HFBA) and perfluorobutanesulfonic acid (PFBS) for 72 h to investigate potential effects on cell proliferation and neoplastic transformation. PFHxA, GenX, PFO2OA, HFBA and PFBS induced no alterations compared to controls at any of the concentrations tested. Exposure to 100 μM PFHxS on the other hand was shown to affect important regulatory cell-cycle proteins (cyclin D1, CDK6, p27, p53 and ERK) and induced cell proliferation, at least in part through activation of the constitutive androstane receptor (CAR) and the peroxisome proliferator-activated receptor alpha (PPARα). PFHxS also altered histone modifications and induced cell malignance by reducing the levels of adhesion proteins (E-cadherin and β-integrin) and promoting cell migration and invasion. These results demonstrate that five out of six alternative PFAS tested are clearly less harmful to MCF-10A cells than previously studied PFOS and PFOA, but raise concerns about PFHxS that also has been associated with breast cancer in epidemiological studies., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Per and polifluorinated substances (PFAS) are ubiquitous and persistent contaminants. Studies have indicated that fetuses and infants can be exposed to these chemicals in utero and through breastfeeding. Despite this, limited data about their effects on brain development are available. Here, we compared the effects of perfluoroctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) exposure in rat primary neurons and neural stem cells (NSC). Treatment with 1-250 μM of either of these compounds caused no effects on cell viability or proliferation in primary neurons, while PFOS exposure increased the NSC proliferation already at the lowest concentration tested (1-100 μM). Further analysis showed that both PFOS and PFOA caused morphological alterations of NSC-derived neurons. The neurons derived from NSC treated with either of the PFAS demonstrated a decrease in cell body area. Exposure to 1 and 10 μM PFOA also affected the neurite network and caused an increase in the number of processes and branches per cell. None of the PFAS caused morphological alterations in primary neurons. These data suggest that NSC, mimicking the immature brain, is clearly more susceptible to PFOS and PFOA exposure than the primary neurons. The PFAS-induced alterations in NSC may be related to neurobehavioral alterations observed in rodents developmentally exposed to these compounds, and show the importance to consider the effects of these compounds on human brain development and disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Underlying drivers of species extinctions need to be better understood for effective conservation of biodiversity. Nearly half of all amphibian species are at risk of extinction, and pollution may be a significant threat as seasonal high-level agrochemical use overlaps with critical windows of larval development. The potential of environmental chemicals to reduce the fitness of future generations may have profound ecological and evolutionary implications. This study characterized effects of male developmental exposure to environmentally relevant concentrations of the anti-androgenic pesticide linuron over two generations of offspring in Xenopus tropicalis frogs. The adult male offspring of pesticide-exposed fathers (F1) showed reduced body size, decreased fertility, and signs of endocrine system disruption. Impacts were further propagated to the grand-offspring (F2), providing evidence of transgenerational effects in amphibians. The adult F2 males demonstrated increased weight and fat body palmitoleic-to-palmitic acid ratio, and decreased plasma glucose levels. The study provides important cross-species evidence of paternal epigenetic inheritance and pollutant-induced transgenerational toxicity, supporting a causal and complex role of environmental contamination in the ongoing species extinctions, particularly of amphibians., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) has been implicated in the etiology of neurodegenerative disorders. BMAA is also a known developmental neurotoxin and research indicates that the sources of human and wildlife exposure may be more diverse than previously anticipated. The aim of the present study was therefore to examine whether BMAA can be transferred into birds' eggs. Egg laying quail were dosed with 14 C-labeled BMAA. The distribution of radioactivity in the birds and their laid eggs was then examined at different time points by autoradiography and phosphoimaging analysis. To evaluate the metabolic stability of the BMAA molecule, the distribution of 14 C-methyl- and 14 C-carboxyl-labeled BMAA were compared. The results revealed a pronounced incorporation of radioactivity in the eggs, predominantly in the yolk but also in the albumen. Imaging analysis showed that the concentrations of radioactivity in the liver decreased about seven times between the 24h and the 72h time points, while the concentrations in egg yolk remained largely unchanged. At 72h the egg yolk contained about five times the concentration of radioactivity in the liver. Both BMAA preparations gave rise to similar distribution pattern in the bird tissues and in the eggs, indicating metabolic stability of the labeled groups. The demonstrated deposition into eggs warrants studies of BMAAs effects on bird development. Moreover, birds' eggs may be a source of human BMAA exposure, provided that the laying birds are exposed to BMAA via their diet., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: Health outcomes, including chronic disease and mortality, attributed to or associated with alcohol abuse are discrepant between African Americans and Whites. To date, the topic is not fully understood and few studies conducted have used biomarker indicators of health. We investigated whether the association between alcohol abuse and biomarkers of the neuroendocrine system vary between black or African American and White respondents aged 34-84 from the Midlife in the United States Study (MIDUS) II (2004-2006) (n=1129). Alcohol abuse was assessed with a modified version of the Michigan Alcohol Screening Test. Ordinary least squared (OLS) regression was used to evaluate whether race moderated the associations between alcohol abuse and four biomarkers-urinary cortisol and serum dehydroepiandrosterone sulfate (DHEA-S), epinephrine and norepinephrine-and two composite summary scores, each consisting of two components that characterize the hypothalamic pituitary adrenal (HPA)-axis and sympathetic nervous systems (SNS), respectively. Covariates included age, sex, education, income, current drinking, smoking, exercise, fast food consumption, heart disease, blood pressure, diabetes, body mass index, medication use, anxiety/depression, sleep duration, and cholesterol markers. Race significantly moderated the associations between alcohol abuse and norepinephrine concentration (χ 2 [1]=4.48, p=0.034) and the SNS composite score (χ 2 [1]=5.83, p=0.016). Alcohol abuse was associated with higher mean norepinephrine levels (b=0.26, standard error (SE)=0.12, p=0.034) and SNS composite score (b=0.23, SE=0.11, p=0.016) for African Americans compared to Whites. Interestingly, for Whites a paradoxical association between alcohol abuse, norepinephrine and SNS levels was observed; those who abused alcohol had lower mean norepinephrine levels than non-abusers. Race differences in neuroendocrine response could be biological pathways that contribute the excess risk of chronic disease and mortality attributed to alcohol abuse among African Americans compared to Whites. Replication of these analyses in larger cohorts are warranted in addition to further studies of underlying mechanisms among Blacks and Whites separately., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The environmental toxin β-N-methylamino-L-alanine (BMAA) has been proposed to contribute to neurodegenerative diseases. We have previously shown that neonatal exposure to BMAA results in dose-dependent cognitive impairments, proteomic alterations and progressive neurodegeneration in the hippocampus of adult rats. A high BMAA dose (460mg/kg) also induced intracellular fibril formation, increased protein ubiquitination and enrichment of proteins important for lipid transport and metabolism. The aim of this study was therefore to elucidate the role of neuronal lipids in BMAA-induced neurodegeneration. By using matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS), we characterized the spatial lipid profile in the hippocampus of six month-old rats that were treated neonatally (postnatal days 9-10) with 460mg/kg BMAA. Multivariate statistical analysis revealed long-term changes in distinct ganglioside species (GM, GD, GT) in the dentate gyrus. These changes could be a consequence of direct effects on ganglioside biosynthesis through the b-series (GM3-GD3-GD2-GD1b-GT1b) and may be linked to astrogliosis. Complementary immunohistochemistry experiments towards GFAP and S100β further verified the role of increased astrocyte activity in BMAA-induced brain damage. This highlights the potential of imaging MS for probing chemical changes associated with neuropathological mechanisms in situ. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The cyanobacterial non-proteinogenic amino acid β-N-methylamino-l-alanine (BMAA) is proposed to be involved in the etiology of amyotrophic lateral sclerosis/parkinsonism dementia complex. When administered as single doses to neonatal rats, BMAA gives rise to cognitive and neurodegenerative impairments in the adult animal. Here, we employed mass spectrometry (LC-MS/MS) and autoradiographic imaging to examine the mother-to-pup transfer of BMAA in rats. The results show that unchanged BMAA was secreted into the milk and distributed to the suckling pups. The concentration of BMAA in pup stomach milk and the neonatal liver peaked after 8h, while the concentration in the pup brain increased throughout the study period. About 1 and 6% of the BMAA recovered from adult liver and brain were released following hydrolysis, suggesting that this fraction was associated with protein. No association to milk protein was observed. Injection of rat pups with [methyl-(14)C]-l-BMAA or [carboxyl-(14)C]-l-BMAA resulted in highly similar distribution patterns, indicating no or low metabolic elimination of the methylamino- or carboxyl groups. In conclusion, BMAA is transported as a free amino acid to rat milk and suckling pups. The results strengthen the proposal that mothers' milk could be a source of exposure for BMAA in human infants., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Introduction: The field of lipid research has made progress and it is now possible to study the lipidome of cells and organelles. A basic requirement of a successful lipid study is adequate pre-analytical sample handling, as some lipids can be unstable and postmortem changes can cause substantial accumulation of free fatty acids (FFAs)., Methods: The aim of the present study was to investigate the effects of conductive heat stabilization and euthanasia methods on FFA levels in the rat brain and liver using liquid chromatography tandem mass spectrometry., Results: The analysis of brain homogenates clearly demonstrated phospholipase activity and time-dependent post-sampling changes in the lipid pool of snap frozen non-stabilized tissue. There was a significant increase in FFAs already at 2min, which continued over time. Heat stabilization was shown to be an efficient method to reduce phospholipase activity and ex vivo lipolysis. Post-sampling effects due to tissue thawing and sample preparation induced a massive release of FFAs (up to 3700%) from non-stabilized liver and brain tissues compared to heat stabilized tissue. Furthermore, the choice of euthanasia method significantly influenced the levels of FFAs in the brain. The FFAs were decreased by 15-44% in the group of animals euthanized by pentobarbital injection compared with CO2 inhalation or decapitation., Discussion: Our results highlight the importance of considering euthanasia methods and pre-analytical treatment in lipid analysis, factors which may otherwise interfere with the outcome of the experiments., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The environmental neurotoxin β-N-methylamino-L-alanine (BMAA) is not an amino acid that is normally found in proteins. Our previous autoradiographic study of (3)H-labeled BMAA in adult mice unexpectedly revealed a tissue distribution similar to that of protein amino acids. The aim of this study was to characterize the distribution of free and protein-bound BMAA in neonatal rat tissues following a short exposure using autoradiographic imaging and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The autoradiographic imaging of (14)C-L-BMAA demonstrated a distinct uptake of radioactivity that was retained following acid extraction in tissues with a high rate of cell turnover and/or protein synthesis. The UHPLC-MS/MS analysis conclusively demonstrated a dose-dependent increase of protein-associated BMAA in neonatal rat tissues. The level of protein-associated BMAA in the liver was more than 10 times higher than that in brain regions not fully protected by the blood-brain barrier which may be due to the higher rate of protein synthesis in the liver. In conclusion, this study demonstrated that BMAA was associated with rat proteins suggesting that BMAA may be misincorporated into proteins. However, protein-associated BMAA seemed to be cleared over time, as none of the samples from adult rats had any detectable free or protein-associated BMAA., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The neurotoxic amino acid β-N-methylamino-l-alanine (BMAA) is produced by most cyanobacteria. BMAA is considered as a potential health threat because of its putative role in neurodegenerative diseases. We have previously observed cognitive disturbances and morphological brain changes in adult rodents exposed to BMAA during the development. The aim of this study was to characterize changes of major intermediary metabolites in serum following neonatal exposure to BMAA using a non-targeted metabolomic approach. NMR spectroscopy was used to obtain serum metabolic profiles from neonatal rats exposed to BMAA (40, 150, 460mg/kg) or vehicle on postnatal days 9-10. Multivariate data analysis of binned NMR data indicated metabolic pattern differences between the different treatment groups. In particular five metabolites, d-glucose, lactate, 3-hydroxybutyrate, creatine and acetate, were changed in serum of BMAA-treated neonatal rats. These metabolites are associated with changes in energy metabolism and amino acid metabolism. Further statistical analysis disclosed that all the identified serum metabolites in the lowest dose group were significantly (p<0.05) decreased. The neonatal rat model used in this study is so far the only animal model that displays significant biochemical and behavioral effects after a low short-term dose of BMAA. The demonstrated perturbation of intermediary metabolism may contribute to BMAA-induced developmental changes that result in long-term effects on adult brain function., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)