Papers of the Month
By David Banks, Deacqunita Diggs, Mahita Kadmiel, Gabriel Knudsen, and Qing Xu
NTP researchers show butter flavoring agents cause respiratory toxicity in rats
National Toxicology Program (NTP) researchers recently demonstrated inhalation exposure to the alpha-diketone flavoring agents, 2,3-butanedione (BD), 2,3-pentanedione (PD), and 2,3-hexanedione (HD), can cause respiratory toxicity in rats. All three agents induced bronchial fibrosis after two weeks of exposure and patchy interstitial fibrosis that resulted in pulmonary function deficits that developed after a two-week recovery.
BD is a naturally occurring alpha-ketone that gives butter its flavor. BD is used as a flavoring agent in food, but it can cause obliterative bronchiolitis when inhaled, which can happen in an occupational setting. PD and HD are being used as substitutes for BD. PD is known to cause significant respiratory toxicity in rats and mice, but until recently HD had not been tested for inhalation toxicity.
NTP scientists found the small structural differences among BD, PD, and HD were sufficient to significantly alter their physical and chemical properties, as well as their reactivity and toxicity after inhalation. Alpha-diketones may exert their toxic effects by enzyme inhibition and protein crosslinking. All three agents inactivated glutathione s-transferase, but HD was significantly less reactive than BD and PD. Similarly, HD exposure resulted in less bronchial fibrosis in these studies. The authors concluded that longer chain alpha-diketones, such as HD, may reduce the likelihood of inhalation exposure and toxicity. (GK)
Morgan DL, Jokinen MP, Johnson CL, Price HC, Gwinn WM, Bousquet RW, Flake GP. 2016. Chemical reactivity and respiratory toxicity of the alpha-diketone flavoring agents: 2,3-butanedione, 2,3-pentanedione, and 2,3-hexanedione. Toxicol Pathol; doi:10.1177/0192623316638962 [Online 29 March 2016].
Tdp2 mediates anticancer drug resistance by processing damaged DNA
NIEHS researchers have revealed how a DNA repair protein, tyrosyl-DNA phosphodiesterase 2 (Tdp2), processes and removes a topoisomerase 2 (Top2) DNA-protein complex induced by damaged DNA. Top2 has been a popular anticancer target because of its crucial role in untangling supercoiled DNA for replication and transcription. Findings from this study helped scientists understand Tdp2-mediated resistance to Top2-targeting drugs and support the use of Tdp2 inhibitors to improve the outcome of cancer treatment. Top2-targeting drugs, such as etoposide, work by trapping Top2 on the damaged DNA to form a DNA-protein complex. Tdp2 removes the complex by cleaving the linkage between the DNA and Top2.
Previous work from the same NIEHS group demonstrated the high-resolution structure of Tdp2 bound with DNA, and this study further dissected the molecular mechanisms underlying the engagement of Tdp2 with damaged DNA. The researchers found that Tdp2 can accommodate DNA lesions with various structures. In addition, the cleavage reaction depends on a single magnesium ion. More importantly, they identified single nucleotide polymorphisms (SNP) in the human Tdp2 gene that blocked the activity of Tdp2. Cells bearing this SNP were more sensitive to etoposide. These results suggest that Tdp2 status may be used as a biomarker in humans to predict individual sensitivity to chemotherapy. (QX)
Schellenberg MJ, Perera L, Strom CN, Waters CA, Monian B, Appel CD, Vilas CK, Williams JG, Ramsden DA, Williams RS. 2016. Reversal of DNA damage induced topoisomerase 2 DNA-protein crosslinks by Tdp2. Nucleic Acids Res 44(8):3829-3844.
Maternal smoking during pregnancy leads to epigenetic changes in newborns
NIEHS scientists, as part of the Pregnancy and Childhood Epigenetics (PACE) consortium, have shed new light on the links between maternal smoking during pregnancy and epigenetic modifications in newborns.
Researchers combined epidemiologic and epigenetic data from 13 different studies and used a statistical method called meta-analysis to determine associations between maternal smoking during pregnancy and genome-wide changes in DNA methylation in newborn blood. Using the Illumina450K platform, researchers measured DNA methylation at more than 450,000 cytosine and guanine nucleotide pairings, or CpG sites, along the DNA sequence to determine the epigenetic modification. More than 6,000 of these CpG sites were differentially methylated relative to maternal smoking during pregnancy. Although many were linked to genes that had already been related to maternal smoking, nearly 3,000 had never been associated with smoking in either children or adults before this study. Some of these novel signals are implicated in the development of smoking-related conditions, such as orofacial cleft and asthma.
The scientists then examined the genes corresponding to these differentially methylated CpG sites. Functional enrichment and methylation transcription analyses were performed to further elucidate the biological processes affected by these epigenetic changes. Anatomical development, phosphate-containing compound metabolism, nervous system development, and embryonic morphogenesis were all implicated. Many of the epigenetic changes were found to continue into later childhood. (DB)
Joubert BR, Felix JF, Yousefi P, Bakulski KM, Just AC, Breton C, Reese S, Markunas CA, Richmond RC, Xu C-J, Kupers LK, Oh SS, Hoyo C, Gruzieva O, Soderhall C, Salas LA, Baiz N, Zhang H, Lepeule J, Ruiz C, Ligthart S, Wang T, Taylor JA, Duijts L, Sharp GC, Jankipersadsing SA, Nilsen RM, Vaez A, Fallin MD, Hu D, Litonjua AA, Fuemmeler BF, Huen K, Kere J, Kull I, Munthe-Kaas MC, Gehring U, Bustamante M, Saurel-Coubizolles MJ, Quraishi BM, Ren J, Tost J, Gonzalez JR, Peters MJ, Haberg SE, Xu Z, van Meurs JB, Gaunt TR, Kerkhof M, Corpeleijn E, Feinberg AP, Eng C, Baccarelli AA, Neelon SEB, Bradman A, Merid K, Bergstrom A, Herceg Z, Hernandez-Vargas H, Brunekreef B, Pinart M, Heude B, Ewart S, Yao J, Lemonnier N, Franco OH Wu MC, Hofman A, McArdle W, Van der Vlies P, Falahi F, Gillman MW, Barcellos LF, Kumar A, Wickman M, Guerra S, Charles M-A, Holloway J, Auffray C, Tiemeier HW, Smith GD, Postma D, Hivert M-F, Eskenazi B, Vrijheid M, Arshad H, Anto JM, Dehghan A, Karmaus W, Annesi-Maesano I, Sunyer J, Ghantous A, Pershagen G, Holland N, Murphy SK, DeMeo DL, Burchard EG, Ladd-Acosta C, Snieder H, Nystad W, Koppelman GH, Relton CL, Jaddoe VWV, Wilcox A, Melen E, London SJ. 2016. DNA methylation in newborns and maternal smoking in pregnancy: genome-wide consortium meta-analysis. Am J Hum Genet 98(4):680-696.
Analysis of bisphenol A and potassium bromate exposure on mouse embryonic fibroblasts
NIEHS researchers used a mouse model of embryonic fibroblast to show that co-exposure of bisphenol A (BPA) with the oxidizing agent potassium bromate altered the cellular microenvironment to promote cell survival by delaying the onset of DNA repair and altering the transcription of DNA repair genes. Suppression of the initiation of base lesion DNA repair is known to promote cell survival after exposure to lesion-inducing agents.
By selecting a cell line deficient in double-strand break repair, the researchers could more precisely examine the cellular responses of base lesion repair pathways to oxidatively induce DNA damage. Cells were dosed with BPA alone, potassium bromate alone, and a combination of both agents. To assess BPA’s influence, DNA lesions, chromatin condensation, transcription profiles, and intracellular pH were measured 4 and 24 hours after exposure.
The study showed that co-exposure of high-dose BPA and potassium bromate generated reactive oxygen species and induced DNA base lesions; however, the cellular response to these lesions was different than the exposure to BPA alone or potassium bromate alone. Co-exposure to BPA altered the microcellular environment, which might have promoted cell survival after combined exposure to a second DNA damaging event. Further studies are needed to determine the effects of BPA on health, especially in combination with other chemicals. (DD)
Gassman NR, Coskun E, Jaruga P, Dizdaroglu M, Wilson SH. 2016. Combined effects of high-dose bisphenol A and oxidizing agent (KBrO3) on cellular microenvironment, gene expression, and chromatin structure of Ku70-deficient mouse embryonic fibroblasts. Environ Health Perspect; doi:10.1289/EHP237 [Online 15 April 2016].
Splenic transcriptome in TTP deficient mice defines the complexity of TTP action
NIEHS researchers used a transcriptomics approach to define the complexity of TTP action in vivo. Tristetraprolin, or TTP, is an mRNA binding protein that acts by binding to certain elements on target mRNAs, enhancing their rate of turnover. TTP deficient mice develop a complex systemic inflammatory syndrome with arthritis and autoimmunity, many aspects of which are mediated by excess production of tumor necrosis factor (TNF). TNF mRNA is a direct TTP target, and the proinflammatory cytokine TNF is overproduced in TTP deficient mice and has many secondary effects of its own.
To remove the secondary effects of TNF action, the researchers performed deep mRNA sequencing of spleens from TTP deficient mice in the setting of TNF receptor deficiency. More than 2,000 transcripts were significantly increased in expression, of which at least 395 contained potential TTP binding sites in their 3’ untranslated regions. However, only a few of these transcripts exhibited discordant effects when the mature mRNA levels were compared with the pre-mRNA, suggesting that many of the observed changes in transcript levels were secondary effects of the TTP deficiency in vivo. These data highlight the complexity of TTP action at the organism level, and point to the need for systematic studies in other cells and tissues to gain increased understanding of TTP functions in vivo. (MK)
Patial S, Stumpo DJ, Young WS 3rd, Ward JM, Flake GP, Blackshear PJ. 2016. Effects of combined tristetraprolin/tumor necrosis factor receptor deficiency on the splenic transcriptome. Mol Cell Biol 36(9):1395-1411.
(David Banks is a postbaccalaureate Intramural Research and Training Award (IRTA) fellow in the NIEHS Intracellular Regulation Group. Deacqunita Diggs, Ph.D., is an Oak Ridge Institute for Science and Education fellow in the U.S. Environmental Protection Agency Developmental Toxicity Branch. Mahita Kadmiel, Ph.D., is a visiting fellow in the NIEHS Molecular Endocrinology Group. Gabriel Knudsen, Ph.D., is a research fellow in the National Cancer Institute, Center for Cancer Research, Laboratory of Toxicology and Toxicokinetics. Qing Xu is a biologist in the NIEHS Metabolism, Genes, and Environment Group.)