Rescuing Alu: recovery of new inserts shows LINE-1 preserves Alu activity through A-tail expansion.

Alu elements are trans-mobilized by the autonomous non-LTR retroelement, LINE-1 (L1). Alu-induced insertion mutagenesis contributes to about 0.1% human genetic disease and is responsible for the majority of the documented instances of human retroelement insertion-induced disease. Here we introduce a SINE recovery method that provides a complementary approach for comprehensive analysis of the impact and biological mechanisms of Alu retrotransposition. Using this approach, we recovered 226 de novo tagged Alu inserts in HeLa cells. Our analysis reveals that in human cells marked Alu inserts driven by either exogenously supplied full length L1 or ORF2 protein are indistinguishable. Four percent of de novo Alu inserts were associated with genomic deletions and rearrangements and lacked the hallmarks of retrotransposition. In contrast to L1 inserts, 5' truncations of Alu inserts are rare, as most of the recovered inserts (96.5%) are full length. De novo Alus show a random pattern of insertion across chromosomes, but further characterization revealed an Alu insertion bias exists favoring insertion near other SINEs, highly conserved elements, with almost 60% landing within genes. De novo Alu inserts show no evidence of RNA editing. Priming for reverse transcription rarely occurred within the first 20 bp (most 5') of the A-tail. The A-tails of recovered inserts show significant expansion, with many at least doubling in length. Sequence manipulation of the construct led to the demonstration that the A-tail expansion likely occurs during insertion due to slippage by the L1 ORF2 protein. We postulate that the A-tail expansion directly impacts Alu evolution by reintroducing new active source elements to counteract the natural loss of active Alus and minimizing Alu extinction.

Molecular reconstruction of extinct LINE-1 elements and their interaction with nonautonomous elements.

Non-long terminal repeat retroelements continue to impact the human genome through cis-activity of long interspersed element-1 (LINE-1 or L1) and trans-mobilization of Alu. Current activity is dominated by modern subfamilies of these elements, leaving behind an evolutionary graveyard of extinct Alu and L1 subfamilies. Because Alu is a nonautonomous element that relies on L1 to retrotranspose, there is the possibility that competition between these elements has driven selection and antagonistic coevolution between Alu and L1. Through analysis of synonymous versus nonsynonymous codon evolution across L1 subfamilies, we find that the C-terminal ORF2 cys domain experienced a dramatic increase in amino acid substitution rate in the transition from L1PA5 to L1PA4 subfamilies. This observation coincides with the previously reported rapid evolution of ORF1 during the same transition period. Ancestral Alu sequences have been previously reconstructed, as their short size and ubiquity have made it relatively easy to retrieve consensus sequences from the human genome. In contrast, creating constructs of extinct L1 copies is a more laborious task. Here, we report our efforts to recreate and evaluate the retrotransposition capabilities of two ancestral L1 elements, L1PA4 and L1PA8 that were active ~18 and ~40 Ma, respectively. Relative to the modern L1PA1 subfamily, we find that both elements are similarly active in a cell culture retrotransposition assay in HeLa, and both are able to efficiently trans-mobilize Alu elements from several subfamilies. Although we observe some variation in Alu subfamily retrotransposition efficiency, any coevolution that may have occurred between LINEs and SINEs is not evident from these data. Population dynamics and stochastic variation in the number of active source elements likely play an important role in individual LINE or SINE subfamily amplification. If coevolution also contributes to changing retrotransposition rates and the progression of subfamilies, cell factors are likely to play an important mediating role in changing LINE-SINE interactions over evolutionary time.

Effects of COREXIT dispersants on cytotoxicity parameters in a cultured human bronchial airway cells, BEAS-2B.

The objective of this study was to assess the cytotoxicity of COREXIT dispersants EC9500A, EC9527A, and EC9580A on human airway BEAS-2B epithelial cells. Cells were exposed to dispersants for 2 or 24 h at concentrations ranging from 0 to 300 ppm. COREXIT EC9527 at 100 ppm produced 50% viability loss as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 24 h. COREXIT 9527 at 200 ppm produced 50% cell death at 2 h and 100% at 24 h. At 300 ppm COREXIT 9527 induced 100% cell death at 2 or 24 h. In the case of COREXIT 9500A 50% cell viability was noted with 200 ppm at 2 or 24 h, with a significant decrease in cell survival to 2% at 300 ppm. In contrast, no marked change in cell viability was observed in cells treated at any COREXIT 9580A concentration examined. Western blot analysis showed an increase in expression of LC3B, a marker of autophagy, in cells treated for 2 h with 300 ppm COREXIT EC9527A as well as 100 or 300 ppm Corexit EC9500A. No marked effect on LC3B expression was observed for any COREXIT 9580A concentration. Apoptosis markers as measured by cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) were detectable only in cells incubated with 300 ppm COREXIT EC9527A. Although all three dispersants induced enhanced generation of reactive oxygen species (ROS) after 2-h treatment at 300 ppm, Western blot analysis revealed that 2-h incubation was not sufficient to induce a significant change in the protein expression of superoxide dismutases SOD1, SOD2, and SOD3. Data thus indicate exposure to certain dispersants may be harmful to human airway epithelial cells in a concentration-dependent manner.

Somatic expression of LINE-1 elements in human tissues.

LINE-1 expression damages host DNA via insertions and endonuclease-dependent DNA double-strand breaks (DSBs) that are highly toxic and mutagenic. The predominant tissue of LINE-1 expression has been considered to be the germ line. We show that both full-length and processed L1 transcripts are widespread in human somatic tissues and transformed cells, with significant variation in both L1 expression and L1 mRNA processing. This is the first demonstration that RNA processing is a major regulator of L1 activity. Many tissues also produce translatable spliced transcript (SpORF2). An Alu retrotransposition assay, COMET assays and 53BP1 foci staining show that the SpORF2 product can support functional ORF2 protein expression and can induce DNA damage in normal cells. Tests of the senescence-associated beta-galactosidase expression suggest that expression of exogenous full-length L1, or the SpORF2 mRNA alone in human fibroblasts and adult stem cells triggers a senescence-like phenotype, which is one of the reported responses to DNA damage. In contrast to previous assumptions that L1 expression is germ line specific, the increased spectrum of tissues exposed to L1-associated damage suggests a role for L1 as an endogenous mutagen in somatic tissues. These findings have potential consequences for the whole organism in the form of cancer and mammalian aging.

The RNA polymerase dictates ORF1 requirement and timing of LINE and SINE retrotransposition.

Mobile elements comprise close to one half of the mass of the human genome. Only LINE-1 (L1), an autonomous non-Long Terminal Repeat (LTR) retrotransposon, and its non-autonomous partners-such as the retropseudogenes, SVA, and the SINE, Alu-are currently active human retroelements. Experimental evidence shows that Alu retrotransposition depends on L1 ORF2 protein, which has led to the presumption that LINEs and SINEs share the same basic insertional mechanism. Our data demonstrate clear differences in the time required to generate insertions between marked Alu and L1 elements. In our tissue culture system, the process of L1 insertion requires close to 48 hours. In contrast to the RNA pol II-driven L1, we find that pol III transcribed elements (Alu, the rodent SINE B2, and the 7SL, U6 and hY sequences) can generate inserts within 24 hours or less. Our analyses demonstrate that the observed retrotransposition timing does not dictate insertion rate and is independent of the type of reporter cassette utilized. The additional time requirement by L1 cannot be directly attributed to differences in transcription, transcript length, splicing processes, ORF2 protein production, or the ability of functional ORF2p to reach the nucleus. However, the insertion rate of a marked Alu transcript drastically drops when driven by an RNA pol II promoter (CMV) and the retrotransposition timing parallels that of L1. Furthermore, the "pol II Alu transcript" behaves like the processed pseudogenes in our retrotransposition assay, requiring supplementation with L1 ORF1p in addition to ORF2p. We postulate that the observed differences in retrotransposition kinetics of these elements are dictated by the type of RNA polymerase generating the transcript. We present a model that highlights the critical differences of LINE and SINE transcripts that likely define their retrotransposition timing.

All y'all need to know 'bout retroelements in cancer.

Genetic instability is one of the principal hallmarks and causative factors in cancer. Human transposable elements (TE) have been reported to cause human diseases, including several types of cancer through insertional mutagenesis of genes critical for preventing or driving malignant transformation. In addition to retrotransposition-associated mutagenesis, TEs have been found to contribute even more genomic rearrangements through non-allelic homologous recombination. TEs also have the potential to generate a wide range of mutations derivation of which is difficult to directly trace to mobile elements, including double strand breaks that may trigger mutagenic genomic rearrangements. Genome-wide hypomethylation of TE promoters and significantly elevated TE expression in almost all human cancers often accompanied by the loss of critical DNA sensing and repair pathways suggests that the negative impact of mobile elements on genome stability should increase as human tumors evolve. The biological consequences of elevated retroelement expression, such as the rate of their amplification, in human cancers remain obscure, particularly, how this increase translates into disease-relevant mutations. This review is focused on the cellular mechanisms that control human TE-associated mutagenesis in cancer and summarizes the current understanding of TE contribution to genetic instability in human malignancies.

ERCC1/XPF limits L1 retrotransposition.

Retrotransposons are currently active in the human and mouse genomes contributing to novel disease mutations and genomic variation via de novo insertions. However, little is known about the interactions of non-long terminal repeat (non-LTR) retrotransposons with the host DNA repair machinery. Based on the model of retrotransposition for the human and mouse LINE-1 element, one likely intermediate is an extension of cDNA that is heterologous to the genomic target, a flap intermediate. To determine whether a human flap endonuclease could recognize and process this potential intermediate, the genetic requirement for the ERCC1/XPF heterodimer during LINE-1 retrotransposition was characterized. Reduction of XPF in human cells increased retrotransposition whereas complementation of ERCC1-deficiency in hamster cells reduced retrotransposition. These results demonstrate for the first time that DNA repair enzymes act to limit non-LTR retrotransposition and may provide insight into the genetic instability phenotypes of ercc1 and xpf individuals.

LINE-1 ORF1 protein enhances Alu SINE retrotransposition.

Retroelements have contributed over one third of the human genome mass. The currently active LINE-1 (L1) codes for two proteins (ORF1p and ORF2p), both strictly required for retrotransposition. In contrast, the non-coding parasitic SINE (Alu) only appears to need the L1 ORF2p for its own amplification. This requirement was previously determined using a tissue culture assay system in human cells (HeLa). Because HeLa are likely to express functional L1 proteins, it is possible that low levels of endogenous ORF1p are necessary for the observed tagged Alu mobilization. By individually expressing ORF1 and ORF2 proteins from both human (L1RP and LRE3) and rodent (L1A102 and L1spa) L1 sources, we demonstrate that increasing amounts of ORF1 expressing vector enhances tagged Alu mobilization in HeLa cells. In addition, using chicken fibroblast cells as an alternate cell culture source, we confirmed that ORF1p is not strictly required for Alu mobilization in our assay. Supporting our observations in HeLa cells, we find that tagged Alu retrotransposition is improved by supplementation of ORF1p in the cultured chicken cells. We postulate that L1 ORF1p plays either a direct or indirect role in enhancing the interaction between the Alu RNA and the required factors needed for its retrotransposition.

Reviving a 60 million year old LINE-1 element.

Mobile elements have significantly impacted genome structure of most organisms. The continued activity of the mobile element, LINE-1 (L1), through time has contributed to the accumulation of over half a million L1 copies in the human genome. Most copies in the human genome belong to evolutionary older extinct L1s. Here we apply our previous published approach to "revive" the extinct L1 PA13A; an L1 family that was active about 60 million year ago (mya). The reconstructed L1PA13A is retrocompentent in culture, but shows a significantly lower level of activity in HeLa cells when compared to the modern L1 element (L1PA1) and a 40 million year old L1PA8. L1 elements code for two proteins (ORF1p and ORF2p) that are necessary for retrotransposition. Using PA13A-PA1 and PA13A-PA8 L1 chimeric elements, we determined that both the ORF1p and ORF2p contribute to the observed decrease in retrotransposition efficiency of L1PA13A. The lower retrotransposition rate of L1PA13A is consistent in both human and rodent cell lines. However, in rodent cells, the chimeric element L1PA:1-13 containing the modern L1PA1 ORF1p shows a recovery in the retrotransposition rate, suggestive that the L1PA13A ORF2p efficiently drives retrotransposition in these cells. The functionality of the L1PA13A ORF2p was further confirmed by demonstrating its ability to drive Alu retrotransposition in rodent cells. The variation in L1PA13A retrotransposition rates observed between rodent and human cells are suggestive that cellular environment significantly affects retrotransposition efficiency, which may be mediated through an interaction with ORF1p. Based on these observations, we speculate that the observed differences between cell lines may reflect an evolutionary adaptation of the L1 element to its host cell.

The role of Alu-derived RNAs in Alzheimer's and other neurodegenerative conditions.

Non-coding RNAs have emerged as essential contributors to neuroinflammation. The Alu element is the most abundant potential source of non-coding RNA in the human genome represented by over 1.1 million copies totaling ∼10% of the genome's mass. Accumulation of "Alu RNA" was observed in the brains of individuals with dementia and Creutzfeldt-Jakob disease - a degenerative brain disorder. "Alu RNAs" activate inflammatory pathways and apoptosis in the non-neural cells. In particular, the "Alu RNA" cytotoxicity is suggested as a mechanism in retinal pigment epithelium (RPE), a compartment damaged in the process of age-related macular degeneration. In RPE cells, the deficiency of Dicer is reported to lead to an accumulation of P3Alu transcripts, subsequent activation of the ERK1/2 signaling pathway, and the formation of NLRP3 inflammasome. In turn, these events result in RPE cell death by apoptosis. Importantly, RPE cells are of neuroectodermal origin, these cells display more similarity to neurons than to other epithelial cells. Thus, it is plausible that the mechanisms of "Alu RNA" cytotoxicity in brain neurons are similar to that in RPE. We hypothesize that accumulation of polymerase III-transcribed noncoding RNA of Alu (P3Alu) may contribute to both neuroinflammation and neurodegeneration associated with Alzheimer's disease (AD) and other degenerative brain disorders. This hypothesis points toward a novel molecular pathway not previously considered for the treatment of AD.

Evaluating different DNA binding domains to modulate L1 ORF2p-driven site-specific retrotransposition events in human cells.

DNA binding domains (DBDs) have been used with great success to impart targeting capabilities to a variety of proteins creating highly useful genomic tools. We evaluated the ability of five types of DBDs and strategies (AAV Rep proteins, Cre, TAL effectors, zinc finger proteins, and Cas9/gRNA system) to target the L1 ORF2 protein to drive retrotransposition of Alu inserts to specific sequences in the human genome. First, we find that the L1 ORF2 protein tolerates the addition of protein domains both at the amino- and carboxy-terminus. Although in some instances retrotransposition efficiencies slightly diminished, all fusion proteins containing an intact ORF2 were capable of driving retrotransposition. Second, the stability of individual ORF2 fusion proteins varies and difficult to predict. Third, DBDs that require the formation of multimers for target recognition are unlikely to modify targeting of ORF2p-driven insertions. Fourth, the more components needed to assemble into a complex to drive targeted retrotransposition, the less likely the strategy will increase targeted insertions. Fifth, abundance of target sequences present in the genome will likely dictate the effectiveness and efficiency of targeted insertions. Lastly, the cleavage capabilities of Cas9 (or a Cas9 nickase variant) are unable to substitute for the L1 ORF2 endonuclease domain functions, suggestive that the endonuclease domain has alternate functions needed for retrotransposition. From these studies, we conclude that the most critical component for the modification of the human L1 ORF2 protein to drive targeted insertions is the selection of the DBD due to the varying functional requirements and impacts on protein stability.

Meeting report for Odd Pols 2016: Ann Arbor 2.0.

The Tenth International Conference on Transcription by RNA Polymerases I, III, IV and V (the 'Odd Pols') was held June 24-28, 2016 at the University of Michigan, Ann Arbor, USA and organized by David Engelke, Deborah Johnson, Richard Maraia, Lawrence Rothblum, David Schneider, Andrzej Wierzbicki and Astrid Engel. The organizers are grateful for the support from the Rackham Graduate School of the University of Michigan for providing the meeting venue. The environment provided a great background with unexpected encounters with fireflies, free live music and a festive fireworks display. The meeting was composed of eleven oral sessions and a poster session. The keynote speaker, Dave Engelke, opened the meeting with his presentation entitled "A personal history of pol III transcription - how we got here from the 'good old days'." The meeting drew attendees from sixteen countries that shared their research discoveries. Here we present some of the highlights from the meeting using summaries provided by the participants.

Carcinogenic effects of oil dispersants: A KEGG pathway-based RNA-seq study of human airway epithelial cells.

The health impacts of the BP oil spill are yet to be further revealed as the toxicological effects of oil products and dispersants on human respiratory system may be latent and complex, and hence difficult to study and follow up. Here we performed RNA-seq analyses of a system of human airway epithelial cells treated with the BP crude oil and/or dispersants Corexit 9500 and Corexit 9527 that were used to help break up the oil spill. Based on the RNA-seq data, we then systemically analyzed the transcriptomic perturbations of the cells at the KEGG pathway level using two pathway-based analysis tools, GAGE (generally applicable gene set enrichment) and GSNCA (Gene Sets Net Correlations Analysis). Our results suggested a pattern of change towards carcinogenesis for the treated cells marked by upregulation of ribosomal biosynthesis (hsa03008) (p=1.97E-13), protein processing (hsa04141) (p=4.09E-7), Wnt signaling (hsa04310) (p=6.76E-3), neurotrophin signaling (hsa04722) (p=7.73E-3) and insulin signaling (hsa04910) (p=1.16E-2) pathways under the dispersant Corexit 9527 treatment, as identified by GAGE analysis. Furthermore, through GSNCA analysis, we identified gene co-expression changes for several KEGG cancer pathways, including small cell lung cancer pathway (hsa05222, p=9.99E-5), under various treatments of oil/dispersant, especially the mixture of oil and Corexit 9527. Overall, our results suggested carcinogenic effects of dispersants (in particular Corexit 9527) and their mixtures with the BP crude oil, and provided further support for more stringent safety precautions and regulations for operations involving long-term respiratory exposure to oil and dispersants.

The Nucleotide Excision Repair Pathway Limits L1 Retrotransposition.

Long interspersed elements 1 (L1) are active mobile elements that constitute almost 17% of the human genome. They amplify through a "copy-and-paste" mechanism termed retrotransposition, and de novo insertions related to these elements have been reported to cause 0.2% of genetic diseases. Our previous data demonstrated that the endonuclease complex ERCC1-XPF, which cleaves a 3' DNA flap structure, limits L1 retrotransposition. Although the ERCC1-XPF endonuclease participates in several different DNA repair pathways, such as single-strand annealing, or in telomere maintenance, its recruitment to DNA lesions is best characterized in the nucleotide excision repair (NER) pathway. To determine if the NER pathway prevents the insertion of retroelements in the genome, we monitored the retrotransposition efficiencies of engineered L1 elements in NER-deficient cells and in their complemented versions. Core proteins of the NER pathway, XPD and XPA, and the lesion binding protein, XPC, are involved in limiting L1 retrotransposition. In addition, sequence analysis of recovered de novo L1 inserts and their genomic locations in NER-deficient cells demonstrated the presence of abnormally large duplications at the site of insertion, suggesting that NER proteins may also play a role in the normal L1 insertion process. Here, we propose new functions for the NER pathway in the maintenance of genome integrity: limitation of insertional mutations caused by retrotransposons and the prevention of potentially mutagenic large genomic duplications at the site of retrotransposon insertion events.

Nickel stimulates L1 retrotransposition by a post-transcriptional mechanism.

Sequence studies of the human genome demonstrate that almost half of the DNA is derived from mobile elements. Most of the current retrotransposition activity arises from L1 and the L1-dependent, non-autonomous elements, such as Alu, contributing to a significant amount of genetic mutation and genomic instability. We present data demonstrating that nickel chloride, but not cobalt chloride, is able to stimulate L1 retrotransposition about 2.5-fold. Our data suggest that the stimulation occurs at a post-transcriptional level, possibly during the integration process. The effect of nickel on the cell is highly complex, limiting the determination of the exact mechanism of this stimulation. The observed stimulation of L1 retrotransposition is not due to a general increase in L1 transcription or an increase in the number of genomic nicks caused by nickel, but more likely caused by a decrease in DNA repair activities that influence the downstream events of retrotransposition. Our observations demonstrate the influence of environmental toxicants on human retroelement activity. We present an additional mechanism for heavy-metal carcinogenesis, where DNA damage through mobile element activation must be considered when dealing with genomic damage/instability in response to environmental agents.

The L1 retrotranspositional stimulation by particulate and soluble cadmium exposure is independent of the generation of DNA breaks.

Human exposure to toxic metals is a concern of the highest priority, due to their vast array of biological effects, including carcinogenicity. The particulate (water insoluble) form of several heavy metals presents a higher carcinogenic potential than its soluble counterparts. Our previous work demonstrates that the particulate forms of different heavy metals, such as nickel oxide, cadmium sulfide and mercury sulfide, stimulate human L1 mobile element activity leading to genomic instability. We present data demonstrating that the soluble form of CdCl2 also stimulates L1 retrotransposition in a dose-dependent manner comparable to the insoluble carcinogenic form of this compound. Reproducible results demonstrated a 2 to 3 fold dose-dependent increase in L1 retrotransposition compared to control cells. Heavy metals may cause DNA breaks through the generation of reactive oxygen species. However, evaluation of DNA damage by comet assay revealed no differences between the negative controls and the CdS-treated cells. In addition, active L1 elements express a protein with endonuclease activity that can generate toxicity through the creation of double strand breaks. To determine the contribution of the L1 endonuclease to the toxicity observed in our metal treatment assays, we compared the wildtype L1 vector with an L1 endonuclease-mutant vector. The presence of an active L1 endonuclease did not contribute significantly to the toxicity observed in any of the CdCl2 or CdS doses evaluated. No correlation between the creation of DNA breaks and L1 activity was observed. Alternatively, heavy metals inhibit enzymatic reactions by displacement of cofactors such as Zn and Mg from enzymes. Concomitant treatment with Mg(Ac)2 and Zn(Ac)2 ppb suppresses the stimulatory effect on L1 activity induced by the 3.8 ppb CdS treatment. Overall, these results are consistent with our previous observations, suggesting that the mechanism of L1 stimulation by heavy metals is most likely due to an overall inhibition of DNA repair proteins or other enzymes caused by the displacement of Mg and Zn from cellular proteins.

SINE Retrotransposition: Evaluation of Alu Activity and Recovery of De Novo Inserts.

Mobile element activity is of great interest due to its impact on genomes. However, the types of mobile elements that inhabit any given genome are remarkably varied. Among the different varieties of mobile elements, the Short Interspersed Elements (SINEs) populate many genomes, including many mammalian species. Although SINEs are parasites of Long Interspersed Elements (LINEs), SINEs have been highly successful in both the primate and rodent genomes. When comparing copy numbers in mammals, SINEs have been vastly more successful than other nonautonomous elements, such as the retropseudogenes and SVA. Interestingly, in the human genome the copy number of Alu (a primate SINE) outnumbers LINE-1 (L1) copies 2 to 1. Estimates suggest that the retrotransposition rate for Alu is tenfold higher than LINE-1 with about 1 insert in every twenty births. Furthermore, Alu-induced mutagenesis is responsible for the majority of the documented instances of human retroelement insertion-induced disease. However, little is known on what contributes to these observed differences between SINEs and LINEs. The development of an assay to monitor SINE retrotransposition in culture has become an important tool for the elucidation of some of these differences. In this chapter, we present details of the SINE retrotransposition assay and the recovery of de novo inserts. We also focus on the nuances that are unique to the SINE assay.

Effects of corexit oil dispersants and the WAF of dispersed oil on DNA damage and repair in cultured human bronchial airway cells, BEAS-2B.

Large quantities of dispersants were used as a method to disperse the roughly 210 million gallons of spilled crude oil that consumed the Gulf of Mexico. Little is known if the oil-dispersant and oil-dispersant mixtures on human airway BEAS-2B epithelial cells. Here we present the cytotoxic and genotoxic in vitro effects on the human lung cells BEAS-2B following exposure to and oil-dispersant mixtures on human airway BEAS-2B epithelial cells. Here we present the cytotoxic and genotoxic in vitro effects on the human lung cells BEAS-2B following exposure to Corexit dispersants EC9500 and EC9527, Water Accommodated Fraction (WAF) -crude, WAF-9500 + Oil, and WAF-9527 + Oil. Cellular cytotoxicity to WAF-dispersed oil samples was observed at concentrations greater than 1000 ppm with over 70% of observed cellular death. At low concentration exposures (100 and 300 ppm) DNA damage was evidenced by the detection of single strand breaks (SSBs) and double strand breaks (DSBs) as measured by alkaline and neutral comet assay analyses. Immunoblot analyses of the phosphorylated histone H2A.X (É£-H2A.X) and tumor suppressor p53 protein confirmed activation of the DNA damage response due to the exposure-induced DNA breaks. Although, many xenobiotics interfere with DNA repair pathways, in vitro evaluation of the nucleotide excision repair (NER) and DSB repair pathways appear to be unaffected by the oil-dispersant mixtures tested. Overall, this study supports that oil-dispersant mixtures induce genotoxic effects in culture.

The impact of oil spill to lung health--Insights from an RNA-seq study of human airway epithelial cells.

The Deepwater Horizon oil spill (BP oil spill) in the Gulf of Mexico was a unique disaster event, where a huge amount of oil spilled from the sea bed and a large volume of dispersants were applied to clean the spill. The operation lasted for almost 3 months and involved >50,000 workers. The potential health hazards to these workers may be significant as previous research suggested an association of persistent respiratory symptoms with exposure to oil and oil dispersants. To reveal the potential effects of oil and oil dispersants on the respiratory system at the molecular level, we evaluated the transcriptomic profile of human airway epithelial cells grown under treatment of crude oil, the dispersants Corexit 9500 and Corexit 9527, and oil-dispersant mixtures. We identified a very strong effect of Corexit 9500 treatment, with 84 genes (response genes) differentially expressed in treatment vs. control samples. We discovered an interactive effect of oil-dispersant mixtures; while no response gene was found for Corexit 9527 treatment alone, cells treated with Corexit 9527+oil mixture showed an increased number of response genes (46 response genes), suggesting a synergic effect of 9527 with oil on airway epithelial cells. Through GO (gene ontology) functional term and pathway-based analysis, we identified upregulation of gene sets involved in angiogenesis and immune responses and downregulation of gene sets involved in cell junctions and steroid synthesis as the prevailing transcriptomic signatures in the cells treated with Corexit 9500, oil, or Corexit 9500+oil mixture. Interestingly, these key molecular signatures coincide with important pathological features observed in common lung diseases, such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. Our study provides mechanistic insights into the detrimental effects of oil and oil dispersants to the respiratory system and suggests significant health impacts of the recent BP oil spill to those people involved in the cleaning operation.

Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes.

Heavy metals such as cadmium, arsenic and nickel are classified as carcinogens. Although the precise mechanism of carcinogenesis is undefined, heavy metal exposure can contribute to genetic damage by inducing double strand breaks (DSBs) as well as inhibiting critical proteins from different DNA repair pathways. Here we take advantage of two previously published culture assay systems developed to address mechanistic aspects of DNA repair to evaluate the effects of heavy metal exposures on competing DNA repair outcomes. Our results demonstrate that exposure to heavy metals significantly alters how cells repair double strand breaks. The effects observed are both specific to the particular metal and dose dependent. Low doses of NiCl2 favored resolution of DSBs through homologous recombination (HR) and single strand annealing (SSA), which were inhibited by higher NiCl2 doses. In contrast, cells exposed to arsenic trioxide preferentially repaired using the "error prone" non-homologous end joining (alt-NHEJ) while inhibiting repair by HR. In addition, we determined that low doses of nickel and cadmium contributed to an increase in mutagenic recombination-mediated by Alu elements, the most numerous family of repetitive elements in humans. Sequence verification confirmed that the majority of the genetic deletions were the result of Alu-mediated non-allelic recombination events that predominantly arose from repair by SSA. All heavy metals showed a shift in the outcomes of alt-NHEJ repair with a significant increase of non-templated sequence insertions at the DSB repair site. Our data suggest that exposure to heavy metals will alter the choice of DNA repair pathway changing the genetic outcome of DSBs repair.