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Papers of the Month

Intramural
By Robin Arnette, Kathleen Foley, Kelly Lenox, Janelle Weaver, and Qing Xu

NTP screens chemicals for liver injury potential

When applied to human cell culture models, a high-throughput transcriptomics platform called TempO-Seq captures biological responses to chemicals that induce liver injury, according to a study by researchers at the National Toxicology Program (NTP) and NIEHS-funded colleagues. The novel approach may better predict the potentially harmful effects of a wide variety of environmental chemicals or pharmaceutical drugs in humans.

Predicting the responses of the human liver to chemical exposures is a major challenge in both pharmaceutical and toxicological research. To address this challenge, the researchers used TempO-Seq to generate high-throughput data for approximately 2,700 human transcripts with highly differentiated in vitro liver models that were exposed to wide-ranging concentrations of 24 reference compounds. This approach readily distinguished liver-injury compounds, such as the chemotherapy drug tamoxifen and pharmaceuticals that were withdrawn from the market (e.g., drug analogues), from compounds rarely associated with liver injury, such as caffeine and sucrose.

Moreover, the authors noted effective modeling of metabolically activated hepatic responses and leverage of the resolving power of concentration-response modeling to identify anticipated biological-response pathways that included hallmarks of liver function. The findings demonstrated that high-throughput transcriptomics, combined with differentiated in vitro liver models, might be an effective tool to model, explore, and interpret toxicological and pharmacological interactions. (JW)

Citation: Ramaiahgari SC, Auerbach SS, Saddler TO, Rice JR, Dunlap PE, Sipes NS, DeVito MJ, Shah RR, Bushel PR, Merrick BA, Paules RS, Ferguson SS. 2019. The power of resolution: contextualized understanding of biological responses to liver injury chemicals using high-throughput transcriptomics and benchmark concentration modeling. Toxicol Sci; doi: 10.1093/toxsci/kfz065 [Online 8 March 2019]. (Story)

Pol B maintains its fidelity in the presence of modified cytosines

The fidelity or precision of DNA polymerase beta (pol B) is maintained when it encounters modified cytosines in the DNA template strand, according to NIEHS scientists. Because pol B adds new nucleotides to DNA as part of the base excision repair (BER) pathway, understanding how the enzyme handles bulky side chains is important to DNA repair.

The researchers made the discovery by studying how cells remove one form of epigenetic tags, called methyl groups, from DNA. The removal process uses ten-eleven translocation enzymes and BER, generating three forms of the oxidized nucleotide cytosine, or 5-methylcytosine (5mC), which are structurally different: 5-hydroxmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). The scientists tested whether these modified forms affected the ability of pol B to incorporate cytosine’s nucleotide partner dGTP into DNA.

Using kinetic assays, the scientists showed that 5mC, 5hmC, and 5fC did not affect the efficiency of pol B to incorporate dGTP, but when 5caC was in the templating position, it reduced pol B’s efficiency 20-fold compared with unmodified cytosine. However, structural studies determined that the backbone of the DNA templating strand shifted approximately 2.5 angstroms to avoid clashing with the carboxy modification of 5caC. This action allowed pol B to do its job, demonstrating that the cytosine epigenetic modifications were accommodated during repair. (RA)

Citation: Howard MJ, Foley KG, Shock DD, Batra VK, Wilson SH. 2019. Molecular basis for the faithful replication of 5-methylcytosine and its oxidized forms by DNA polymerase beta. J Biol Chem; doi: 10.1074/jbc.RA118.006809 [Online 18 March 2019].

Aberrant estrogen signaling induces ovarian disorder

NIEHS researchers have revealed that dysfunction of estrogen signaling may induce polycystic ovary syndrome (PCOS), a common reproductive problem associated with hormone imbalance and ovulation failure in women. The findings provide new insights into the molecular mechanisms for PCOS and infertility.

PCOS is considered a disorder of the hypothalamic-pituitary-gonadal (HPG) axis, which is tightly controlled by a regulation feedback loop. In the loop, hypothalamus hormones trigger the pituitary gland to release luteinizing hormone (LH), which stimulates the ovary to produce estrogen that inhibits the secretion of hypothalamic hormones. Previous studies showed that estrogen receptor alpha (ERalpha) is required for HPG regulation, because global ERalpha knockout mice (ERKO) display elevated LH and phenotypes of PCOS. In this study, the researchers developed a novel mouse model (global ERalpha-null knockout, or PitERtgKO) in which pituitary ERalpha was re-expressed in the ERKO mice to investigate the role of pituitary ERalpha in the development of PCOS.

They found that PitERtgKO mice exhibited a more severe cystic ovarian phenotype than their ERKO littermates, although LH in PitERtgKO was restored to a lower level found in wild-type mice. They further determined that sporadic secretion of LH mediated by pituitary ERalpha, rather than the persistently high LH, caused the severe phenotypes of PCOS. (QX)

Citation: Arao Y, Hamilton KJ, Wu SP, Tsai MJ, DeMayo FJ, Korach, KS. 2019. Dysregulation of hypothalamic-pituitary estrogen receptor alpha-mediated signaling causes episodic LH secretion and cystic ovary. FASEB J; doi: 10.1096/fj.201802653RR [Online 13 March 2019].

Shift work linked to epigenetic changes

NIEHS scientists reported associations between shift work and changes in DNA methylation patterns that are consistent with long-term health effects. The new study is the first to evaluate associations between epigenetic age acceleration and a history of shift work.

Using methylation data for 2,574 women enrolled in the Sister Study, the team used established epigenetic clocks to identify women whose estimated epigenetic age was older than their chronological age, also known as epigenetic age acceleration. The researchers also measured DNA methylation across the genome. They found that the difference between epigenetic age and chronological age was, on average, three years greater for women who worked for ten or more years in jobs involving night shifts compared with women who never worked night shifts.

Shift work overall, or working irregular hours or rotating shifts, was most strongly correlated with methylation levels at a certain site in the Ataxin 7 gene, which is linked with susceptibility to and prognosis of tumors. Night shift work was also associated with methylation levels at a site within the circadian rhythm gene ZFHX3, which may function as a tumor suppressor. These methylated regions may play a role in the known association between shift work and breast cancer. (KL)

Citation: White AJ, Kresovich JK, Xu Z, Sandler DP, Taylor JA. 2019. Shift work, DNA methylation and epigenetic age. Int J Epidemiol; doi: 10.1093.ije/dyz027 [Online 15 March 2019].

Phthalates, oxidative stress, and inflammation in pregnant women

A team led by NIEHS researchers found that phthalate and phthalate alternative metabolites have associations with the oxidative stress marker 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha) that arises from different pathways. The scientists used a novel approach to distinguish whether 8-iso-PGF2alpha arose through inflammation or oxidative stress. They assert that knowing this information will aid in determining the overall toxicological effects of phthalates on human health and may also help design strategies for mitigating the effects of exposure.

Phthalates are used to soften plastics, but some phthalates have been associated with birth defects. The mechanisms for these relationships are unknown but could involve inflammation and oxidative stress.

Researchers measured 8-iso-PGF2alpha and examined associations with phthalate and phthalate alternative levels in urine samples from pregnant women in The Infant Development and the Environment Study (TIDES). Other NIEHS scientists discovered that both oxidative stress and inflammation could produce 8-iso-PGF2alpha, and that amounts from each pathway could be calculated using the ratio of 8-iso-PGF2alpha to prostaglandin-PGF2alpha (PGF2alpha). In the TIDES study, the researchers observed associations between most phthalate and phthalate alternative metabolites and 8-iso-PGF2alpha. Using the 8-iso-PGF2alpha/PGF2alpha ratio, they also observed that, although most phthalates correlated with the oxidative stress pathway, a specific subgroup of phthalates with similar structures had associations with the inflammation pathway. (KF)

Citation: van’t Erve TJ, Rosen EM, Barrett ES, Nguyen RHN, Sathyanarayana S, Milne GL, Calafat AM, Swan SH, Ferguson KK. 2019. Phthalates and phthalate alternatives have diverse associations with oxidative stress and inflammation in pregnant women. Environ Sci Technol 53(6):3258–3267.

(Kathleen Foley is an Intramural Research Training Award postbaccalaureate fellow in the NIEHS Receptor Biology Group. Janelle Weaver, Ph.D., is a contract writer for the NIEHS Office of Communications and Public Liaison. Qing Xu is a biologist in the NIEHS Metabolism, Genes, and Environment Group.)