Validation of Salamander Dermal Mucus Swabs as a Novel, Nonlethal Approach for Amphibian Metabolomics and Glutathione Analysis Following Pesticide Exposure.

Evaluating biomarkers of stress in amphibians is critical to conservation, yet current techniques are often destructive and/or time-consuming, which limits ease of use. In the present study, we validate the use of dermal swabs in spotted salamanders (Ambystoma maculatum) for biochemical profiling, as well as glutathione (GSH) stress response following pesticide exposure. Thirty-three purchased spotted salamanders were acclimated to laboratory conditions at Washington College (Chestertown, MD, USA) for 4 weeks. Following acclimation, salamanders were randomly sorted into three groups for an 8-h pesticide exposure on soil: control with no pesticide, 2,4-dichlorophenoxyacetic acid (2,4-D), or chlorpyrifos. Before and after exposure, mucus samples were obtained by gently rubbing a polyester-tipped swab 50 times across the ventral and dorsal surfaces. Salamanders were humanely euthanized and dissected to remove the brain for acetylcholinesterase and liver for GSH and hepatic metabolome analyses, and a whole-body tissue homogenate was used for pesticide quantification. Levels of GSH were present in lower quantities on dermal swabs relative to liver tissues for chlorpyrifos, 2,4-D, and control treatments. However, 2,4-D exposures demonstrated a large effect size increase for GSH levels in livers (Cohen's d = 0.925, p = 0.036). Other GSH increases were statistically insignificant, and effect sizes were characterized as small for 2,4-D mucosal swabs (d = 0.36), medium for chlorpyrifos mucosal swabs (d = 0.713), and negligible for chlorpyrifos liver levels (d = 0.012). The metabolomics analyses indicated that the urea cycle, alanine, and glutamate metabolism biological pathways were perturbed by both sets of pesticide exposures. Obtaining mucus samples through dermal swabbing in amphibians is a viable technique for evaluating health in these imperiled taxa. Environ Toxicol Chem 2024;43:1126-1137. © 2024 SETAC.

Inactivation of yeast Isw2 chromatin remodeling enzyme mimics longevity effect of calorie restriction via induction of genotoxic stress response.

ATP-dependent chromatin remodeling is involved in all DNA transactions and is linked to numerous human diseases. We explored functions of chromatin remodelers during cellular aging. Deletion of ISW2, or mutations inactivating the Isw2 enzyme complex, extends yeast replicative lifespan. This extension by ISW2 deletion is epistatic to the longevity effect of calorie restriction (CR), and this mechanism is distinct from suppression of TOR signaling by CR. Transcriptome analysis indicates that isw2Δ partially mimics an upregulated stress response in CR cells. In particular, isw2Δ cells show an increased response to genotoxic stresses, and the DNA repair enzyme Rad51 is important for isw2Δ-mediated longevity. We show that lifespan is also extended in C. elegans by reducing levels of athp-2, a putative ortholog of Itc1/ACF1, a critical subunit of the enzyme complex. Our findings demonstrate that the ISWI class of ATP-dependent chromatin remodeling complexes plays a conserved role during aging and in CR.

Telomere stability and carcinogenesis: an off-again, on-again relationship.

Previous studies in mice have demonstrated antagonistic effects of telomerase loss on carcinogenesis. Telomere attrition can promote genome instability, thereby stimulating initiation of early-stage cancers, but can also inhibit tumorigenesis by promoting permanent cell growth arrest or death. Human cancers likely develop in cell lineages with low levels of telomerase, leading to telomere losses in early lesions, followed by subsequent activation of telomerase. Mouse models constitutively lacking telomerase have thus not addressed how telomere losses within telomerase-proficient cells have an impact on carcinogenesis. Using a novel transgenic mouse model, Begus-Nahrmann et al. demonstrate in this issue of the JCI that transient telomere dysfunction in telomerase-proficient animals is a potent stimulus of tumor formation.

The effect of genetic background on the function of Saccharomyces cerevisiae mlh1 alleles that correspond to HNPCC missense mutations.

Germline mutations in the DNA mismatch repair (MMR) gene MLH1 are associated with a large percentage of hereditary non-polyposis colorectal cancers. There are approximately 250 known human mutations in MLH1. Of these, one-third are missense variants that are often difficult to characterize with regards to pathogenicity. We analysed 28 alleles of baker's yeast MLH1 that correspond to non-truncating human mutant alleles listed in online HNPCC databases, 13 of which had not been previously studied in functional assays. Using the highly sensitive lys2::InsE-A(14) reversion rate assay, we determined the MMR proficiency conferred by each allele in the S288c strain of Saccharomyces cerevisiae. Seven alleles conferred a null phenotype for MMR and eight others showed significant MMR defects, suggesting that all 15 are likely to be pathogenic in humans. In addition, we observed a strong correlation between these results, limited results from previous functional assays and clinical data. To test whether the potential pathogenicity of certain alleles depends on the genetic background of the host, we examined the mutation rates conferred by the mlh1 alleles in a second yeast strain, SK1, which is approximately 0.7% divergent from S288c. Many alleles displayed a difference in MMR efficiency between strain backgrounds with decreasing differences as the severity of the MMR defect increased. These findings suggest that genetic background can play an important role in determining the pathogenicity of MMR alleles and may explain cases of atypical colorectal cancer inheritance.