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Papers of the month

Intramural
By Rachel Carroll, Anika Dzierlenga, Salahuddin Syed, Heather Vellers, and Qing Xu

NTP optimizes liver model for improved toxicity screening

National Toxicology Program scientists have recently developed a novel way of culturing liver cells to vastly improve modeling of the human liver. Because the liver plays a vital role in how the body processes and eliminates drugs and other chemicals, models that reflect its complex physiology are in high demand. In this new process, three-dimensional (3D) HepaRG liver cells were grown in spheroid structures that more closely resemble the architecture of the human liver. This approach will allow for more effective screening of chemicals with the potential to cause liver toxicity.

Although liver models have improved substantially in recent years, they fail to reliably predict liver toxicity due to various factors, including poor differentiation, limited lifespan in cell culture, and accurate representation of the expression and function of drug metabolizing enzymes. This new 3D HepaRG spheroid model was tested rigorously for these parameters. It was found to be a cost-effective and user-friendly way to achieve consistency and maintain the cultured cells for a toxicity screening.

The model also maintains drug metabolizing activity, which is key for predicting metabolism-dependent toxicities. Finally, this model accurately reflected the liver toxicity of several chemicals that are known to cause liver toxicity in humans. For example, it distinguished liver-toxic troglitazone and trovafloxacin from the safer analogues rosiglitazone and levofloxacin, respectively. (AD)

Citation: Ramaiahgari SC, Waidyanatha S, Dixon D, DeVito MJ, Paules RS, Ferguson SS. 2017. From the Cover: Three-dimensional (3D) HepRG spheroid model with physiologically relevant xenobiotic metabolism competence and hepatocyte functionality for liver toxicity screening. Toxicol Sci 159(1):124–136.

Observing DNA polymerase mu repair DNA breaks

NIEHS researchers have uncovered the molecular characteristics and dynamic structural changes that enable nucleic acid synthesis by polymerase mu (pol mu) during DNA double-strand break (DSB) repair. The findings offer insight into the unique role of pol mu in repairing damaged DNA and maintaining the integrity of the genome.

Pol mu inserts nucleotides to fill gaps at DSBs in DNA during physiological processes ranging from the nonhomologous end joining DSB repair pathway, to the processes that generate antibody diversity. In this study, the researchers used a technique called time-lapse x-ray crystallography to obtain real-time atomic level snapshots of pol mu undergoing DNA synthesis. As a result, the scientists were able to visualize the detailed molecular features of the enzymatic reaction mediated by pol mu.

The researchers tracked the structural changes of pol mu before, during, and after nucleotide insertion. They identified a new metal ion that transiently associates with the active site and stabilizes the product complex. Together with dynamic structural changes in the active site, they hypothesized that this metal ion plays a crucial role during DSBs. Consistent with previous observations, pol mu displayed only limited structure conversion during DNA synthesis, supporting its capability to accommodate more diverse damaged ends and substrates compared with other polymerases. (QX)

Citation: Jamsen JA, Beard WA, Pedersen LC, Shock DD, Moon AF, Krahn JM, Bebenek K, Kunkel TA, Wilson SH. 2017. Time-lapse crystallography snapshots of a double-strand break repair polymerase in action. Nat Commun 8(1):253.

House dust increases white blood cell counts

Using data from the National Health and Nutrition Examination Survey (NHANES) and the Agricultural Lung Health Study (ALHS), NIEHS scientists and their collaborators are the first to report an association between house dust endotoxin concentration and blood leukocyte count. Taken together with previous studies of experimental human endotoxin inhalation, the finding suggests that the number of leukocytes in the circulation of healthy adults may be determined, in part, by long-term residential exposure to endotoxin.

Endotoxin, an inflammatory lipid found on the exterior of Gram-negative bacteria, is released when bacteria are disrupted. Leukocytes, or white blood cells, patrol the bloodstream for pathogens. Blood leukocyte count is known to increase acutely during infection, but researchers were unsure whether the number of circulating leukocytes in uninfected people was affected by long-term residential exposure to endotoxin.

In both the NHANES and ALHS studies, endotoxin concentration was measured in bed and bedroom floor dust samples. Statistical analyses revealed a positive association between endotoxin concentration and the total count of circulating leukocytes. Study participants with a specific genetic variant of the immune receptor that detects endotoxin, TLR4, had no such increase in leukocytes with endotoxin, suggesting that the endotoxin effect is genetically controlled. (HV)

Citation: Fessler MB, Carnes MU, Salo PM, Wilkerson J, Cohn RD, King D, Hoppin JA, Sandler DP, Travlos G, London SJ, Thorne PS, Zeldin DC. 2017. House dust endotoxin and peripheral leukocyte counts: results from two large epidemiologic studies. Environ Health Perspect 125(5):057010.

Pol mu remains unchanged during NHEJ

NIEHS scientists and their collaborators determined that the catalytic structure of family X-member DNA polymerase mu (pol mu) remains unchanged during binding and incorporation of ribonucleotides. Pol mu participates in repair of DNA double-strand breaks (DSBs) during nonhomologous end joining (NHEJ) and poorly discriminates against ribonucleotides.

To examine how pol mu incorporates ribonucleotide triphosphates (rNTPs), the investigators used x-ray crystallography to examine structures of pre- and postcatalytic complexes of pol mu with a ribonucleotide bound at the active site. The results revealed that pol mu binds and incorporates an rNTP with normal active site geometry and no distortion of the DNA substrate or nucleotide. In addition, when comparing rNTP incorporation by wild-type pol mu with that of mutant pol mu, they found synergistic interactions with multiple active site residues that were different from those in polymerases with greater discrimination.

Taken together, these findings are consistent with the hypothesis that rNTP incorporation by pol mu can be advantageous in gap-filling synthesis during DSB repair by NHEJ, particularly in nonreplicating cells containing a high ratio of ribonucleotides to deoxyribonucleotides. (HV)

Citation: Moon AF, Pryor JM, Ramsden DA, Kunkel TA, Bebenek K, Pedersen LC. 2017. Structural accommodation of ribonucleotide incorporation by the DNA repair enzyme polymerase mu. Nucleic Acids Res 45(15):9138–9148.

ZATT reverses DNA-protein crosslinks during chemotherapy

In a paper published in the journal Science, NIEHS researchers and their collaborators identified a new protein named ZATT that works together with another protein called TDP2 to remove DNA-protein cross-links (DPCs). This research is important, because a class of front-line chemotherapeutic drugs hijacks a cellular enzyme and causes it to trap a DNA break inside of a DPC. This type of DNA damage is challenging for cancer cells to repair and is responsible for the potent cell-killing effects of these drugs.

The human genome becomes entangled when compacted into a cell nucleus, and an enzyme called topoisomerase 2 (TOP2) is needed to untangle DNA, especially in rapidly growing tumor cells. Environmental toxins, antibiotics, and commonly used chemotherapy agents prevent TOP2 from completing its reaction cycle. More importantly, these compounds trap an intermediate state of TOP2 that contains a DNA break with ends that are crosslinked to the inactivated TOP2.

In this study, the scientists were able to show that TDP2 and ZATT first remove the DPC, allowing cellular DNA repair machinery to re-seal the DNA break. Chemotherapy has many side effects and can fail if tumors develop resistance. Understanding how cells respond to and fix trapped TOP2 using TDP2 and ZATT is important for developing better treatment strategies or developing new co-therapies to improve treatment outcomes. (SS)

Citation: Schellenberg MJ, Lieberman JA, Herrero-Ruiz A, Butler LR, Williams JG, Munoz-Cabello AM, Mueller GA, London RE, Cortes-Ledesma F, Williams RS. 2017. ZATT (ZNF451)-mediated resolution of topoisomerase 2 DNA-protein cross-links. Science 357(6358):1412−1416. (Story)

(Rachel Carroll, Ph.D., is a research fellow in the NIEHS Biostatistics and Computational Biology Branch. Anika Dzierlenga, Ph.D., is an Intramural Research Training Award (IRTA) fellow in the NIEHS Developmental and Reproductive Toxicology Group. Salahuddin Syed, Ph.D., is an IRTA fellow in the NIEHS DNA Replication Fidelity Group. Heather Vellers, Ph.D., is an IRTA fellow in the NIEHS Environmental Genetics Group. Qing Xu is a biologist in the NIEHS Metabolism, Genes, and Environment Group.)