Papers of the Month
By Janelle Weaver
Rat oral toxicity studies can produce inconsistent results
The reproducibility of studies that evaluate rodent oral toxicity resulting from short-term chemical exposure is lower than expected, according to researchers from the NIEHS Division of the National Toxicology Program.
Regulatory agencies currently rely on oral toxicity data in rodents to determine hazard categorization, assign appropriate labeling to alert consumers, and perform risk assessments. However, the field of toxicology is developing approaches that do not require the use of laboratory animals. To set expectations regarding the performance of these new methods, it is necessary to develop a reliable reference dataset to characterize the limitations and variability of animal toxicity studies.
To meet this need, the researchers compiled and analyzed rat toxicity data for 2,441 chemicals. For hazard classification, they examined the dose level expected to result in 50% lethality after a single oral administration of each substance. On average, replicate studies produced the same hazard categorization for a given chemical only 60% of the time. Additional analyses suggested that the low reproducibility of the results was likely due to inherent biological or protocol variability. According to the authors, this margin of uncertainty can be leveraged to provide important context for animal-free approaches.
Citation: Karmaus AL, Mansouri K, To KT, Blake B, Fitzpatrick J, Strickland J, Patlewicz G, Allen D, Casey W, Kleinstreuer N. 2022. Evaluation of variability across rat acute oral systemic toxicity studies. Toxicol Sci 188(1):34–47.
Avoiding phthalates could prevent preterm births
Phthalate exposure during pregnancy may be a preventable risk factor for preterm delivery, according to NIEHS researchers and their collaborators.
Phthalates are synthetic chemicals used in everyday consumer products such as personal care items and food processing and packaging. As a result, phthalate exposure is widespread among pregnant individuals. But the evidence has been mixed regarding the potential link between phthalate exposure and preterm birth, which affects approximately 10% of pregnancies in the United States and represents a leading cause of neonatal mortality and illness.
To address this issue, the researchers analyzed 11 phthalate metabolites in urine samples collected from 6,045 pregnant women who participated in 16 studies in the U.S. during the past four decades. According to the authors, the study represents the largest prospective investigation of phthalate exposure in pregnancy and preterm birth.
Higher concentrations for several prevalent phthalates were associated with greater odds of delivering preterm. In particular, an increased risk was posed by mono-N-butyl phthalate, mono-isobutyl phthalate, mono(2-ethyl-5-carboxypentyl) phthalate, and mono(3-carboxypropyl) phthalate.
Additional analyses suggested that hypothetical interventions to reduce exposure to multiple phthalates could produce a significant decrease in preterm births. As noted by the authors, these findings highlight the need for public health and policy measures to reduce phthalate exposures among pregnant individuals.
Citation: Welch BM, Keil AP, Buckley JP, Calafat AM, Christenbury KE, Engel SM, O'Brien KM, Rosen EM, James-Todd T, Zota AR, Ferguson KK, Pooled Phthalate Exposure and Preterm Birth Study Group; Alshawabkeh AN, Cordero JF, Meeker JD, Barrett ES, Bush NR, Nguyen RHN, Sathyanarayana S, Swan SH, Cantonwine DE, McElrath TF, Aalborg J, Dabelea D, Starling AP, Hauser R, Messerlian C, Zhang Y, Bradman A, Eskenazi B, Harley KG, Holland N, Bloom MS, Newman RB, Wenzel AG, Braun JM, Lanphear BP, Yolton K, Factor-Litvak P, Herbstman JB, Rauh VA, Drobnis EZ, Sparks AE, Redmon JB, Wang C, Binder AM, Michels KB, Baird DD, Jukic AMZ, Weinberg CR, Wilcox AJ, Rich DQ, Weinberger B, Padmanabhan V, Watkins DJ, Hertz-Picciotto I, Schmidt RJ. 2022. Associations between prenatal urinary biomarkers of phthalate exposure and preterm birth: a pooled study of 16 US cohorts. JAMA Pediatr 11:e222252.
Structures shed light on DNA repair process
In-depth analysis of crystal structures revealed how an enzyme called polymerase lambda helps when both strands in the DNA double helix are severed, according to NIEHS researchers and their collaborators.
Genomic integrity exists under imminent threat of breakage from environmental exposure to ionizing radiation. The most toxic form of genomic damage occurs with DNA double-strand breaks (DSBs), whose persistence can lead to cancers and other diseases in humans. DSBs can be repaired through the non-homologous end-joining pathway (NHEJ), which efficiently prevents or minimizes sequence loss. During this process, polymerase lambda fills sequence gaps of DSB substrates.
The researchers examined X-ray crystal structures to better understand how polymerase lambda engages its preferred DSB substrates. Their analysis allowed for a breakdown of the enzyme’s active center into its most critical components.
The results revealed that key structural features and hydrogen-bonding networks work in concert to simultaneously stabilize both sides of the tenuous, potentially toxic DNA break during NHEJ. In addition, the findings highlight the important role played by a structural feature called the thumb loop, which is unique to polymerase lambda. According to the authors, such information presents a firm foundation for future investigation of DSB repair through NHEJ.
Citation: Kaminski AM, Chiruvella KK, Ramsden DA, Bebenek K, Kunkel TA, Pedersen LC.. 2022. Analysis of diverse double-strand break synapsis with Polλ reveals basis for unique substrate specificity in nonhomologous end-joining. Nat Commun 13(1):3806.
Two hormones ensure normal testis formation in mice
The proper maintenance of testis structures in mouse embryos depends on hormones called anti-Müllerian hormone (AMH) and activin B, according to NIEHS scientists and their collaborators.
The researchers discovered that these two hormones act together to maintain the identity of Sertoli cells. These testicular cells are known to play a critical role in testis formation and sperm production. The appearance of Sertoli cells in the embryonic testis is thought to be controlled exclusively by transcription factors within the cells. To their surprise, the group in the Reproductive and Developmental Biology Laboratory found that AMH and activin B, both are hormones and not transcription factors, were necessary to maintain the testis program in Sertoli cells.
The absence of these Sertoli cell-derived hormones led to partial sex reversal and resulted in the formation of ovotestes — gonads with features of both testes and ovaries. The ovotestes remained to adulthood and produced both sperm and egg cells, an intriguing phenomenon mostly found in non-mammalian species such as fish.
According to the authors, the findings could explain so-called freemartin effects in pregnant sheep and cows carrying XY and XX twins, which were described by Greek scholar Marcus Terentius Varro in 27 BC. In these cases, the XX twin develops testis structures under the influence of an XY twin. Specifically, AMH and activin B produced by the XY embryo might act as freemartin factors that can masculinize the fetal ovary of the XX twin, rendering the XX twin infertile. In addition, the study may also provide insight into the mechanisms behind disorders of sex development in humans.
Citation: Rodriguez KF, Brown PR, Amato CM, Nicol B, Liu CF, Xu X, Yao HH. 2022. Somatic cell fate maintenance in mouse fetal testes via autocrine/paracrine action of AMH and activin B. Nat Commun 13(1):4130.
How coronaviruses evade immune sensors
A new study shows how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, degrades viral molecules to avoid detection by host defense systems, according to NIEHS researchers and their collaborators.
SARS-CoV-2 has infected millions of people worldwide and led to the ongoing pandemic. Despite the rapid development of effective vaccines, new treatments are urgently needed due to emerging SARS-CoV-2 variants and the prevalence of breakthrough infections among those already vaccinated.
One promising antiviral target is a non-structural protein called Nsp15. Coronaviruses produce and deploy this enzyme to cleave viral RNA molecules, preferentially targeting the nucleoside uridine, thereby evading detection by host immune sensors. Yet Nsp15 remains one of the most understudied non-structural proteins, and it has not been clear how the enzyme recognizes and processes double-stranded RNA.
To address this knowledge gap, the researchers used cryogenic electron microscopy to solve the structures of SARS-CoV-2 bound to double-stranded RNA. The results showed that Nsp15 processes double-stranded RNA using a unique base-flipping mechanism. Specifically, the engaged uridine flips out from the double-stranded RNA helix and is positioned in the active site for cleavage. According to the authors, this work reveals multiple RNA-protein interfaces that could be targeted for structure-based drug design.
Citation: Frazier MN, Wilson IM, Krahn JM, Butay KJ, Dillard LB, Borgnia MJ, Stanley RE. 2022. Flipped over U: structural basis for dsRNA cleavage by the SARS-CoV-2 endoribonuclease. Nucleic Acids Res 50(14):8290–8301.