Global DNA Adenine Methylation in Caenorhabditis elegans after Multigenerational Exposure to Silver Nanoparticles and Silver Nitrate.

Multigenerational and transgenerational reproductive toxicity in a model nematode Caenorhabditis elegans has been shown previously after exposure to silver nanoparticles (Ag-NPs) and silver ions (AgNO3). However, there is a limited understanding on the transfer mechanism of the increased reproductive sensitivity to subsequent generations. This study examines changes in DNA methylation at epigenetic mark N6-methyl-2'-deoxyadenosine (6mdA) after multigenerational exposure of C. elegans to pristine and transformed-via-sulfidation Ag-NPs and AgNO3. Levels of 6mdA were measured as 6mdA/dA ratios prior to C. elegans exposure (F0) after two generations of exposure (F2) and two generations of rescue (F4) using high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). Although both AgNO3 and Ag-NPs induced multigenerational reproductive toxicity, only AgNO3 exposure caused a significant increase in global 6mdA levels after exposures (F2). However, after two generations of rescue (F4), the 6mdA levels in AgNO3 treatment returned to F0 levels, suggesting other epigenetic modifications may be also involved. No significant changes in global DNA methylation levels were observed after exposure to pristine and sulfidized sAg-NPs. This study demonstrates the involvement of an epigenetic mark in AgNO3 reproductive toxicity and suggests that AgNO3 and Ag-NPs may have different toxicity mechanisms.

Epigenetic effects induced by silver nanoparticles in Caenorhabditis elegans after multigenerational exposure.

Multigenerational effects of silver nanoparticles (Ag-NPs) on reproduction of the soil nematode Caenorhabditis elegans have been observed previously. However, mechanisms of this reproductive sensitivity are unknown. Here we examine whether epigenetic changes occur as a result of multigenerational exposure to Ag-NPs and whether such modifications can be inherited by unexposed generations. Changes at histone methylation markers, histone 3 lysine 4 dimethylation (H3K4me2) and histone 3 lysine 9 trimethylation (H3K9me3), known to affect reproduction, as well as changes in the expression of the genes encoding demethylases and methyltransferases associated with the selected markers, were investigated. We exposed C. elegans at EC30 to AgNO3, pristine Ag-NPs, and its environmentally transformed product, sulfidized Ag-NPs (sAg-NPs). Histone methylation levels at H3K4me2 increase in response to pristine Ag-NP exposure and did not recover after rescue from the exposure, suggesting transgenerational inheritance. Compared to pristine Ag-NPs, exposure to transformed sAg-NPs significantly decreased H3K4me2 and H3K9me3 levels. These changes in the histone methylation were also supported by expression of spr-5 and jmjd-2 (H3K4me2 and H3K9me3 demethylases, respectively) and set-30 (H3K4me2 methyltransferase). Our study demonstrates that multigenerational exposure to Ag-NPs induces epigenetic changes that are inherited by unexposed offspring. However, environmental transformations of Ag-NPs may also reduce toxicity via epigenetic mechanisms, such as changes at histone methylation.

Genomic mutations after multigenerational exposure of Caenorhabditis elegans to pristine and sulfidized silver nanoparticles.

Our previous study showed heritable reproductive toxicity in the nematode Caenorhabditis elegans after multigenerational exposure to AgNO3 and silver nanoparticles (Ag-NPs). The aim of this study was to determine whether such inheritable effects are correlated with induced germline mutations in C. elegans. Individual C. elegans lineages were exposed for 10 generations to equitoxic concentrations at EC30 of AgNO3, Ag-NPs, and sulfidized Ag-NPs (sAg-NPs), a predominant environmentally transformed product of pristine Ag-NPs. The mutations were detected via whole genome DNA sequencing approach by comparing F0 and F10 generations. An increase in the total number of variants, though not statistically significant, was observed for all Ag treatments and the variants were mainly contributed by single nucleotide polymorphisms (SNPs). This potentially contributed towards reproductive as well as growth toxicity shown previously after ten generations of exposure in every Ag treatment. However, despite Ag-NPs and AgNO3 inducing stronger reproductive toxicity than sAg-NPs, exposure to sAg-NPs resulted in higher mutation accumulation with significant increase in the number of transversions. Thus our results suggest that other mechanisms of inheritance, such as epigenetics, may be at play in Ag-NP- and AgNO3-induced multigenerational and transgenerational reproductive toxicity.

Multigenerational exposure to silver ions and silver nanoparticles reveals heightened sensitivity and epigenetic memory in Caenorhabditis elegans.

The effects from multigenerational exposures to engineered nanoparticles (ENPs) in their pristine and transformed states are currently unknown despite such exposures being an increasingly common scenario in natural environments. Here, we examine how exposure over 10 generations affects the sensitivity of the nematode Caenorhabditis elegans to pristine and sulfidized Ag ENPs and AgNO3 We also include populations that were initially exposed over six generations but kept unexposed for subsequent four generations to allow recovery from exposure. Toxicity of the different silver forms decreased in the order AgNO3, Ag ENPs and Ag2S ENPs. Continuous exposure to Ag ENPs and AgNO3 caused pronounced sensitization (approx. 10-fold) in the F2 generation, which was sustained until F10. This sensitization was less pronounced for Ag2S ENP exposures, indicating different toxicity mechanisms. Subtle changes in size and lifespan were also measured. In the recovery populations, the sensitivity to Ag ENPs and AgNO3 resulting from the initial multigenerational exposure persisted. Their response sensitivity for all endpoints was most closely related to the last ancestral exposed generation (F5), rather than unexposed controls. The mechanisms of transgenerational transfer of sensitivity are probably organized through the epigenome, and we encourage others to investigate such effects as a priority for mechanistic toxicology.

Identification, Recombinant Expression, and Biochemical Analysis of Putative Secondary Product Glucosyltransferases from Citrus paradisi.

Flavonoid and limonoid glycosides influence taste properties as well as marketability of Citrus fruit and products, particularly grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semiconserved PSPG box motif, SMART-RACE RT-PCR, and primer walking to full-length coding regions; screening a directionally cloned young grapefruit leaf EST library; designing primers against sequences from other Citrus species; or identifying PGTs from Citrus contigs in the harvEST database. The PGT proteins associated with the identified full-length coding regions were recombinantly expressed in Escherichia coli and/or Pichia pastoris and then tested for activity with a suite of substrates including flavonoid, simple phenolic, coumarin, and/or limonoid compounds. A number of these compounds were eliminated from the predicted and/or potential substrate pool for the identified PGTs. Enzyme activity was detected in some instances with quercetin and catechol glucosyltransferase activities having been identified.