Transgenerational bone toxicity in F3 medaka (Oryzias latipes) induced by ancestral benzo[a]pyrene exposure: Cellular and transcriptomic insights.

Benzo[a]pyrene (BaP), a ubiquitous pollutant, raises environmental health concerns due to induction of bone toxicity in the unexposed offspring. Exposure of F0 ancestor medaka (Oryzias latipes) to 1 µg/L BaP for 21 days causes reduced vertebral bone thickness in the unexposed F3 male offspring. To reveal the inherited modifications, osteoblast (OB) abundance and molecular signaling pathways of transgenerational BaP-induced bone thinning were assessed. Histomorphometric analysis showed a reduction in OB abundance. Analyses of the miRNA and mRNA transcriptomes revealed the dysregulation of Wnt signaling (frzb/ola-miR-1-3p, sfrp5/ola-miR-96-5p/miR-455-5p) and bone morphogenetic protein (Bmp) signaling (bmp3/ola-miR-96-5p/miR-181b-5p/miR-199a-5p/miR-205-5p/miR-455-5p). Both pathways are major indicators of impaired bone formation, while the altered Rank signaling in osteoclasts (c-fos/miR-205-5p) suggests a potentially augmented bone resorption. Interestingly, a typical BaP-responsive pathway, the Nrf2-mediated oxidative stress response (gst/ola-miR-181b-5p/miR-199a-5p/miR-205), was also affected. Moreover, mRNA levels of epigenetic modification enzymes (e.g., hdac6, hdac7, kdm5b) were found dysregulated. The findings indicated that epigenetic factors (e.g., miRNAs, histone modifications) may directly regulate the expression of genes associated with transgenerational BaP bone toxicity and warrants further studies. The identified candidate genes and miRNAs may serve as potential biomarkers for BaP-induced bone disease and as indicators of historic exposures in wild fish for conservation purposes.

Multigenerational Impacts of Benzo[a]pyrene on Bone Modeling and Remodeling in Medaka (Oryzias latipes).

Ancestral benzo[a]pyrene (BaP) (1 µg/L, 21 days) exposure has previously been shown to cause skeletal deformities in medaka (Oryzias latipes) larvae in the F1-F3 generation. However, when and how this deformity is induced during bone development remain to be elucidated. The col10a1:nlGFP/osx:mCherry double transgenic medaka model was employed to determine the temporal and spatial changes of col10a1:nlGFP- positive osteochondral progenitor cells (OPCs) and osx:mCherry-positive premature osteoblasts (POBs) [8 days postfertilization (dpf)-31 dpf] in combination with changes in bone mineralization at the tissue level. Ancestral BaP exposure delayed the development of col10a1:nlGFP- and osx:mCherry-positive osteoblasts and reduced the abundance of col10a1:nlGFP-positive osteoblast progenitors and col10a1:nlGFP/osx:mCherry double-positive premature osteoblasts during critical windows of early vertebral bone formation, associated with reduced bone mineralization in embryos (14 dpf) and larvae (31 dpf), compressed vertebral segments in larvae (31 dpf), and reduced bone thickness in adult male medaka (6 months old) of the F1-F3 generations. Both Col10a1:nlGFP and osx:mCherry were identified as potential targets of epigenetic modifications underlying the transgenerational inheritance of BaP bone toxicity. The present study provides novel knowledge of the underlying mechanisms of transgenerational toxicity of BaP at the cellular level.

Hypoxia Causes Transgenerational Impairment of Ovarian Development and Hatching Success in Fish.

Hypoxia is a pressing environmental problem in both marine and freshwater ecosystems globally, and this problem will be further exacerbated by global warming in the coming decades. Recently, we reported that hypoxia can cause transgenerational impairment of sperm quality and quantity in fish (in F0, F1, and F2 generations) through DNA methylome modifications. Here, we provide evidence that female fish ( Oryzias melastigma) exposed to hypoxia exhibit reproductive impairments (follicle atresia and retarded oocyte development), leading to a drastic reduction in hatching success in the F2 generation of the transgenerational group, although they have never been exposed to hypoxia. Further analyses show that the observed transgenerational impairments in ovarian functions are related to changes in the DNA methylation and expression pattern of two gene clusters that are closely associated with stress-induced cell cycle arrest and cell apoptosis. The observed epigenetic and transgenerational alterations suggest that hypoxia may pose a significant threat to the sustainability of natural fish populations.

Transcriptomic analysis reveals transgenerational effect of hypoxia on the neural control of testicular functions.

There are over 400 hypoxic zones in the ocean worldwide. Both laboratory and field studies have shown that hypoxia causes endocrine disruption and reproductive impairments in vertebrates. More importantly, our recent study discovered that parental (F0) hypoxia exposure resulted in the transgenerational impairment of sperm quality in the F2 generation through the epigenetic regulation of germ cells. In the present study, we aim to test the hypothesis that the brain, as the major regulator of the brain-pituitary-gonad (BPG) axis, is also involved in the observed transgenerational effect. Using comparative transcriptomic analysis on brain tissues of marine medaka Oryzias melastigma, 45 common differentially expressed genes caused by parental hypoxia exposure were found in the hypoxic group of the F0 and F2 generations, and the transgenerational groups of the F2 generation. The bioinformatic analysis on this deregulated gene cluster further highlighted the possible involvement of the brain in the transgenerational effect of hypoxia on testicular structure, including abnormal morphologies of the epididymis and the seminal vesicle, and degeneration of the seminiferous tubule. This finding is concordant to the result of hematoxylin and eosin staining, which showed the reduction of testicular lobular diameter in the F0 and F2 generations. Our study demonstrated for the first time the involvement of the brain in the transgenerational effect of hypoxia.

Ancestral benzo[a]pyrene exposure affects bone integrity in F3 adult fish (Oryzias latipes).

Benzo[a]pyrene (BaP) at an environmentally relevant concentration (1µg/L) has previously been shown to affect bone development in a transgenerational manner in F3 medaka (Oryzias latipes) larvae (17dph). Here, we provide novel histomorphometric data demonstrating that the impaired bone formation at an early life stage is not recoverable and can result in a persistent transgenerational impairment of bone metabolism in F3 adult fish. A decrease in bone thickness and the occurrence of microcracks in ancestrally BaP-treated adult male fish (F3) were revealed by MicroCt measurement and histopathological analysis. The expression of twenty conserved bone miRNAs were screened in medaka and their relative expression (in the F3 ancestral BaP treatment vs the F3 control fish) were determined by quantitative real-time PCR. Attempt was made to link bone miRNA expression with the potential target bone mRNA expression in medaka. Five functional pairs of mRNA/miRNA were identified (Osx/miR-214, Col2a1b/miR-29b, Runx2/miR-204, Sox9b/miR-199a-3p, APC/miR-27b). Unique knowledge of bone-related miRNA expression in medaka in response to ancestral BaP-exposure in the F3 generation is presented. From the ecological risk assessment perspective, BaP needs to be regarded as a transgenerational skeletal toxicant which exerts a far-reaching impact on fish survival and fitness. Given that the underlying mechanisms of cartilage/bone formation are conserved between medaka and mammals, the results may also shed light on the potential transgenerational effect of BaP on skeletal disorders in mammals/humans.