Papers of the Month
By Nicholas Alagna, Mimi Huang, Sanya Mehta, Arif Rahman, Dahea You
New NTP 5-day assay speeds toxicity screening of chemicals
NTP scientists evaluated a high-throughput transcriptomics approach using liver and kidney tissue from five-day assays in male rats to estimate the toxicological potency of chemicals. Toxicity and carcinogenicity are typically assessed by the resource intensive two-year cancer bioassay. In the five-day assays, the authors determined toxicological potency based on the most sensitive sets of genes active in liver and kidney. For most chemicals, the results approximated the toxicological potency derived from the most sensitive histopathological effects — independent of target tissue or organ — observed in male rats in long-term assays. Notably, these approximations were similar in female rats, as well as in male and female mice. The findings suggest that estimates of transcriptomics-based potency from short-term in vivo assays can, in the absence of other data, provide a rapid and effective estimate of toxicological potency.
Motivated by the need for alternative and more rapid and efficient assays, the authors used benchmark dose (BMD) analysis to compare toxicological potency between short- and long-term in vivo assays. Regulatory agencies such as the U.S. Environmental Protection Agency use the lower bound estimate of the BMD as the preferred metric for calculating chemical exposure levels that may pose minimal human risk. (AR)
Citation: Gwinn WM, Auerbach SS, Parham F, Stout MD, Waidyanatha S, Mutlu E, Collins B, Paules RS, Merrick BA, Ferguson S, Ramaiahgari S, Bucher JR, Sparrow B, Toy H, Gorospe J, Machesky N, Shah RR, Balik-Meisner MR, Mav D, Phadke DP, Roberts G, DeVito MJ. 2020. Evaluation of 5-day in vivo rat liver and kidney with high-throughput transcriptomics for estimating benchmark doses of apical outcomes. Toxicol Sci; doi: 10.1093/toxsci/kfaa081 [Online 3 June 2020].
CLP1 is a critical negative regulator of tRNA processing
NIEHS researchers showed that an enzyme called CLP1 plays an important role in transfer RNA (tRNA) processing by regulating the ligation of tRNAs. They also demonstrated that mature, functional tRNAs are generated from pre-tRNAs through a process called TSEN, or (tRNA splicing endonuclease)-mediated splicing of introns. Mutations in CLP1 and the TSEN complex often lead to severe neurological disorders.
Using a technique that allowed Escherichia coli to produce several proteins at once, the scientists expressed and reconstituted the TSEN protein complex, which was able to cleave tRNA. TSEN complex alone was sufficient for removing tRNA introns but CLP1, a binding partner for TSEN, was needed to correctly regulate the ligation step that generates mature tRNAs and tRNA intronic circular RNAs (tricRNAs). Genetic knockdown of CLP1 led to increases in mature tRNAs and tricRNAs, suggesting that CLP1 acts as a negative modulator of tRNA processing. Based on these observations, the authors suggest that disruption of tRNA processing may cause distinct subtypes of neurodegenerative disease, such as pontocerebellar hypoplasia that occurs when CLP1 or TSEN is mutated.
Taken together, this study expanded our knowledge of tRNA splicing regulation and established a recombinant system that generates biochemically active TSEN complex useful for future studies. (DY)
Citation: Hayne CK, Schmidt CA, Haque MI, Matera AG, Stanley RE. 2020. Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage. Nucleic Acids Res; doi: 10.1093/nar/gkaa438 [Online 1 June 2020].
Interneuronal alpha7 nAChRs serve function in theta wave production
Alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) on the surface of interneurons, which are a type of neuron, are involved in the production of electrical oscillations known as theta waves, according to NIEHS researchers and their collaborators. Theta waves, which arise from the hippocampus and other regions, are involved in several crucial cognitive functions and processes, such as spatial learning and memory and navigation. The team previously showed that muscarinic acetylcholine receptors (mAChRs) facilitated the generation of theta oscillations in physically active mice, but also observed results that implicated nAChRs in their regulation.
To explore the role nAChRs play in hippocampal theta oscillations, the researchers delivered methylylcaconitine (MLA), a selective antagonist of alpha7 nAChRs, into the hippocampi of live mice. They observed a significant decrease in the power of theta waves in running mice, indicating that the inhibited receptors contribute to theta wave production. Additionally, by knocking out expression of alpha7 nAChRs in a set of neuronal subtypes, the group determined which subpopulation of neurons expressing alpha7 nAChRs is responsible for theta power regulation. They found that theta power was significantly weakened in mice with alpha7 nAChRs knockout in interneurons, especially in a type called oriens lacunosum moleculare interneurons. (NA)
Citation: Gu Z, Smith KG, Alexander GM, Guerreiro I, Dudek SM, Gutkin B, Jensen P, Yakel JL. 2020. Hippocampal interneuronal alpha7 nAChRs modulate theta oscillations in freely moving mice. Cell Rep 31(10):107740.
A new tool predicts chemical effects on the human body
Researchers at NIEHS and the National Toxicology Program developed the Tox21BodyMap to predict which organs in the human body may be affected by a chemical. The tool will help scientists generate novel hypotheses to test, prioritize chemicals for toxicity testing, and identify knowledge gaps.
To identify organs that could potentially be affected by a chemical, Tox21BodyMap uses data from 971 high-throughput screening assays that evaluated approximately 10,000 unique chemicals. Specifically, it combines information about which gene an assay targets, how highly expressed that gene is in a human organ, and at what tested concentrations a chemical generated a positive assay result. The result is an overall picture of chemical bioactivity. For instance, estradiol is active in multiple assays that target the estrogen receptor. Organs where these receptors are highly expressed, and thus likely to be targeted by estradiol exposure, are the female urogenital system, vagina, uterus, and breast tissue. In addition to allowing users to customize assay sensitivity and organ gene expression specificity, Tox21BodyMap provides multiple visualizations of the data, highlighting target organs on a map of the body, as well as showing a web of network connections and providing downloadable data. (MH)
Citation: Borrel A, Auerbach SS, Houck KA, Kleinstreuer NC. 2020. Tox21BodyMap: a webtool to map chemical effects on the human body. Nucleic Acids Res 48(W1):W472–W476.
Loss of APE2 is a key vulnerability in BRCA1/2 deficient cancer cells
NIEHS researchers, as part of an international team, determined that the protein APE2 is important for resolving topoisomerase 1-generated blocked 3' DNA ends. The scientists knew that loss of APE2 in breast cancer cells with mutated BRCA1 and BRCA2 was lethal, but they did not understand the mechanism behind it. The work may lead to the development of therapeutic agents against these types of tumors.
As tumor suppressor genes, BRCA1 and BRCA2 encode proteins that regulate processes which prevent DNA damage. Particularly, BRCA1/2 proteins are important for facilitating homologous recombination repair of lesions that prevent DNA synthesis. Through genome-wide interaction studies combined with structural and biochemical analysis of APE2, the researchers demonstrated that APE2 acts as a nuclease, or a type of protein that functions like a pair of molecular scissors. In BRCA1/2 deficient tumors, APE2 is critical for cutting 3' DNA blocks that arise endogenously from natural body processes involving topoisomerase 1 and serves as an essential backup to BRCA-mediated DNA repair.
The findings highlight 3' DNA blocks as a major vulnerability in tumor cells that are deficient in homologous recombination due to their BRCA1/2 mutation. Development of an APE2 inhibitor to treat BRCA1/2-mediated breast cancers may be a promising personalized medicine approach. (SM)
Citation: Alvarez-Quilon A, Wojtaszek JL, Mathieu MC, Patel T, Appel CD, Hustedt N, Rossi SE, Wallace BD, Setiaputra D, Adam S, Ohashi Y, Melo H, Cho T, Gervais C, Munoz IM, Grazzini E, Young JTF, Rouse J, Zinda M, Williams RS, Durocher D. 2020. Endogenous DNA 3' blocks are vulnerabilities for BRCA1 and BRCA2 deficiency and are reversed by the APE2 nuclease. Mol Cell 78(6):1152–1165.e8. (Story)
(Nicholas Alagna is an Intramural Research Training Award [IRTA] fellow in the NIEHS Mechanisms of Mutation Group. Mimi Huang, Ph.D., is an IRTA fellow in the Division of the National Toxicology Program (DNTP) Systems Toxicology Group. Sanya Mehta is an IRTA postbaccalaureate fellow in the NIEHS Matrix Biology Group. Arif Rahman, Ph.D., is a visiting fellow in the DNTP Toxicoinformatics Group. Dahea You, Pharm.D., Ph.D., is an IRTA postdoctoral fellow in the DNTP Molecular Toxicology and Genomics Group.)