Dual chemical labeling enables nucleotide-resolution mapping of DNA abasic sites and common alkylation damage in human mitochondrial DNA.

Mitochondrial DNA (mtDNA) modifications play an emerging role in innate immunity and inflammatory diseases. Nonetheless, relatively little is known regarding the locations of mtDNA modifications. Such information is critically important for deciphering their roles in mtDNA instability, mtDNA-mediated immune and inflammatory responses, and mitochondrial disorders. The affinity probe-based enrichment of lesion-containing DNA represents a key strategy for sequencing DNA modifications. Existing methods are limited in the enrichment specificity of abasic (AP) sites, a prevalent DNA modification and repair intermediate. Herein, we devise a novel approach, termed dual chemical labeling-assisted sequencing (DCL-seq), for mapping AP sites. DCL-seq features two designer compounds for enriching and mapping AP sites specifically at single-nucleotide resolution. For proof of principle, we mapped AP sites in mtDNA from HeLa cells under different biological conditions. The resulting AP site maps coincide with mtDNA regions with low TFAM (mitochondrial transcription factor A) coverage and with potential G-quadruplex-forming sequences. In addition, we demonstrated the broader applicability of the method in sequencing other DNA modifications in mtDNA, such as N7-methyl-2'-deoxyguanosine and N3-methyl-2'-deoxyadenosine, when coupled with a lesion-specific repair enzyme. Together, DCL-seq holds the promise to sequence multiple DNA modifications in various biological samples.

Revealing the toxicity of monovalent and trivalent thallium to medaka fish in controlled exposure conditions.

Thallium (Tl) is a rare earth element increasingly being used in high-technology manufacturing. It is also an emerging pollutant with high exposure and toxicity risks to aquatic ecosystems. Tl exists in the environment in a monovalent [thallous, Tl(I)] or trivalent [thallic, Tl(III)] state. Currently, the stability of the two Tl species in natural water is uncertain and the toxicity in algae and daphnia are inconsistent due to lack of robust characterization of Tl species and matrix effects, while studies with fish are sparse. In this study, larvae of medaka fish (Oryzias latipes) were dosed with environmentally relevant concentrations of Tl(I) or Tl(III) spiked into synthetic and natural river water for 7 days to observe the toxic effects of two Tl species on fish. The transformation of Tl(I) and Tl(III) in water was analyzed by high performance liquid chromatography coupled with inductively coupled plasma and mass spectrometry. Analytical and toxicity results showed that Tl(I) is more stable presenting higher mortality and bioconcentration in medaka than Tl(III) in different water matrices. Tl(I)-induced LC50 and body burden in treated fish were highly correlated with its competitive ion, potassium (K), especially in waters containing medium K levels. This study provides reliable evidence regarding the stability and toxicity of Tl(I) and Tl(III) as well as the interaction of aqueous K versus Tl(I) in fish. Such information is useful for justifying water-quality guidelines and ecological risks of Tl pollution in natural water ecosystems.

Differential alteration in reproductive toxicity of medaka fish on exposure to nanoscale zerovalent iron and its oxidation products.

Nanoscale zerovalent iron (nZVI) is a redox-active nanomaterial commonly used in remediation of soil and groundwater pollution and wastewater treatment processes. A large quantity of nZVI (e.g., >100 mg/L) accidentally released from in situ sites to nearby oxygenized aquifers could be rapidly oxidized to iron oxides (e.g., Fe3O4 or Fe2O3) and ions (e.g., Fe2+), for acute hypoxia effects to aquatic life. However, we do not know the ecotoxicological fate of nZVI and its oxidation products at lower, environmentally concentrations in surface water receiving waterborne transportation or effluent discharge in terms of exposure to aquatic vertebrate species. This study assessed the causal effect on reproductive toxicity in medaka adults (Oryzias latipes) of carboxymethyl cellulose-stabilized nZVI (CMC-nZVI), Fe2+ and iron oxide nanoparticles (nFe3O4) with 21-day aqueous exposure at 5 and 20 mg/L (Fe-equivalent). Such concentrations did not significantly change the dissolved oxygen, oxidation-reduction potential or pH values in the 3 iron solutions during the fish exposure period. Neither CMC-nZVI nor Fe2+ treated adults showed altered daily egg production (fecundity) and oxidative stress responses in observed tissues, as compared to controls. However, the fecundity in nFe3O4 (20 mg/L)-treated pairs was significantly decreased, with increased incidence of abnormal immature oocytes in the ovary. As well, nFe3O4 treatment suppressed activities of the antioxidants superoxide dismutase and expression of glutathione peroxidase (gpx) in the brain and ovary. Although nFe3O4 or Fe2+ treatments inhibited mRNA expression of hepatic estrogen receptor (er-α) in females, plasma levels of sex hormones and (Na, K)-ATPase activity in gills of treated fish did not differ from controls for both sexes. Hence, oxidation products (e.g., nFe3O4) from nZVI at lower milligram-per-liter levels may be potent in inducing nanoparticle-specific reproductive toxicity in medaka fish by inducing oxidative stress in female gonads. MAIN FINDING: nZVI oxidation product nFe3O4 at lower mg/L induces nanoparticle-specific reproductive toxicity in medaka fish.

Illicit drug ketamine induces adverse effects from behavioral alterations and oxidative stress to p53-regulated apoptosis in medaka fish under environmentally relevant exposures.

With increasing problems of drug abuse worldwide, aquatic ecosystems are contaminated by human pharmaceuticals from the discharge of hospital or municipal effluent. However, ecotoxicity data and related toxic mechanism for neuroactive controlled or illicit drugs are still lacking, so assessing the associated hazardous risk is difficult. This study aims to investigate the behavioral changes, oxidative stress, gene expression and neurotoxic or apoptosis effect(s) in larvae of medaka fish (Oryzias latipes) with environmentally relevant exposures of ketamine (KET) solutions for 1-14 days. KET exposure at an environmentally relevant concentration (0.004 µM) to 40 µM conferred specific patterns in larval swimming behavior during 24 h. At 14 days, such exposure induced dose- and/or time-dependent alteration on reactive oxygen species induction, the activity of antioxidants catalase and superoxide dismutase, glutathione S-transferase and malondialdehyde contents in fish bodies. KET-induced oxidative stress disrupted the expression of acetylcholinesterase and p53-regulated apoptosis pathways and increased caspase expression in medaka larvae. The toxic responses of medaka larvae, in terms of chemical effects, were qualitatively analogous to those of zebrafish and mammals. Our results implicate a toxicological impact of waterborne KET on fish development and human health, for potential ecological risks of directly releasing neuroactive drugs-containing wastewater into the aquatic environment.