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Environmental Factor, July 2015

Extramural papers of the month

By Nancy Lemontagne

Monitoring gene mutation using low volume blood samples

An NIEHS Small Business Innovative Research grantee and colleagues developed a simple method for quickly detecting red blood cells with mutant forms of the PIG-A gene in 100-microliter blood samples. Mutations in the PIG-A gene are an in vivo indicator of mutation and can be simply and rapidly quantified using flow cytometry. The new assay should prove useful for DNA damage research, including studies of environmental factors that modify normal rates of mutation.

The researchers extended an approach they previously developed in rodent models to analyze reticulocytes and erythrocytes in human blood. To test the approach, they processed and analyzed three independent blood samples from 52 healthy, nonsmoking adults. They found that the frequency of PIG-A mutant reticulocytes and erythrocytes increased with donor age. Replicate samples showed little variability in the average PIG-A mutant frequency — about 10 percent for reticulocytes and 2 percent for erythrocytes. However, the researchers observed a thirtyfold range in reticulocyte PIG-A mutation frequency between different donors. They also found that people generally had higher frequencies of PIG-A mutant blood cells than rodents.

The researchers say that this new technique should prove valuable for various biomonitoring applications, including the study of accidental chemical or radiation exposures, occupational exposures, drugs undergoing clinical trials, cancer therapy treatments, and population-based epidemiology studies of environmental exposures.

Citation Dertinger SD, Avlasevich SL, Bemis JC, Chen Y, MacGregor JT . 2015. Human erythrocyte PIG-A assay: an easily monitored index of gene mutation requiring low volume blood samples. Environ Mol Mutagen 56(4):366-377. 

3-D cell culture provides model to study toxicant effects on endocervix

An NIEHS grantee and colleagues are pioneering a method for growing endocervical tissue on scaffolds, creating a 3-D tissue model that can function and interact normally with hormones and other organs. The new model will allow the study of infection and fertility within endocervix tissue in a more physiological way, including how toxicants, drugs, vaccines, and pathogens affect the tissue.

The endocervix tissue lines the cervical canal between the uterus and the vagina, helping to regulate the delicate balance between the tolerance necessary for conception and the protection necessary to keep out pathogens. To create a functional 3-D endocervix tissue culture, the researchers grew human endocervical cells on highly porous polystyrene scaffolds. They used a mixed population of cells that included epithelial and stromal cells.

The researchers assessed how the cultured cells responded to fluctuating levels of the reproductive hormones estradiol and progesterone that mimicked a 28-day menstrual cycle. The cells appeared to function similarly to normal female reproductive organs in that they expressed estrogen and progesterone receptors, proliferated, responded to hormones, produced mucus, and secreted cytokines and growth factors.

Citation Arslan SY, Yu Y, Burdette JE, Pavone ME, Hope TJ, Woodruff TK, Kim JJ . 2015. Novel three dimensional human endocervix cultures respond to 28-day hormone treatment. Endocrinology 156(4):1602-1609.

Faster assessment of carcinogenic potential for PAH mixtures

An NIEHS grantee and colleagues have developed a faster, more accurate method to assess cancer risk from exposure to polycyclic aromatic hydrocarbons (PAHs). People are primarily exposed to PAHs in the form of mixtures, and this proof-of-concept study demonstrates a first step towards moving away from risk assessments based on individual components of PAH mixtures, to using methods that examine the whole mixture.

To determine the carcinogenic risk of PAH mixtures, the researchers measured the chemical bioactivity profile in skin cells of mice just after short-term PAH exposure. The bioactivity profile, which provides a unique fingerprint of genes and pathways activated by chemicals after exposure, can be used for predicting long-term consequences such as cancer. They tested PAH mixtures found in coal tar, diesel exhaust, and cigarette smoke. After only 12 hours, the researchers could predict the ability of certain PAH mixtures to cause cancer. Other methods take months for tumors to develop.

Although the method needs further testing, the findings demonstrate that long-term cancer outcome for PAH mixtures can be predicted by evaluating bioactivity after short-term exposure. Since the bioactivity profile provides gene signatures that are tied to chemical mechanisms of action, this information could also provide insight into alternate mechanisms of PAH carcinogenesis and related mechanisms for complex mixtures.

Citation Tilton SC, Siddens LK, Krueger SK, Larkin AJ, Lohr CV, Williams DE, Baird WM, Waters KM . 2015. Mechanism-based classification of PAH mixtures to predict carcinogenic potential. Toxicol Sci 146(1):135-145.

Th17 cells convert into regulatory T cells during immune response

New research, funded in part by NIEHS, shows that the Th17 lineage of T helper cells, which can cause severe human inflammatory diseases, can also differentiate into regulatory T cells to help resolve inflammation. The instability and plasticity of Th17 might offer a new therapeutic target for inflammatory diseases.

The body’s inflammatory response to infection is beneficial, but unregulated inflammation can contribute to inflammatory diseases such as rheumatoid arthritis, psoriasis, and multiple sclerosis. Studies have found that Th17 cells show instability, as they can stop expressing their signature inflammatory cytokine IL-17A. They also show plasticity by expressing cytokines typical of other T-cells. To find out if TH17 cells are merely changing expression of a few cytokines or physiologically undergoing a global genetic reprogramming, the researchers developed new fate-mapping mouse models to track Th17 cells during immune responses.

They found that CD4+ T cells that formerly expressed IL-17A go on to acquire an anti-inflammatory phenotype. During an immune response, the TH17 cells showed a change in their signature transcriptional profile and acquired a strong regulatory capacity. The transcriptional profiles of Th17 cells, before and after conversion into regulatory T cells, also revealed a role for transforming growth factor-beta signaling and the aryl hydrocarbon receptor in the conversion process.

The researchers say that the TH17 cell plasticity might be used to develop new and more effective therapies that restore immune tolerance in chronic inflammatory or autoimmune diseases, without the negative side-effects of some systemic immunosuppressive therapies.

Citation Gagliani N, Vesely MC, Iseppon A, Brockmann L, Xu H, Palm NW, de Zoete MR, Licona-Limon P, Paiva RS, Ching T, Weaver C, Zi X, Pan X, Fan R, Garmire LX, Cotton MJ, Drier Y, Bernstein B, Geginat J, Stockinger B, Esplugues E, Huber S, Flavell RA . 2015. Th17 cells transdifferentiate into regulatory T cells during resolution of inflammation. Nature; doi:10.1038/nature14452 [Online 29 April 2015].

(Nancy Lamontagne is a science writer with MDB Inc., a contractor for the NIEHS Division of Extramural Research and Training.)

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