Drew R. Peterson

Modulation of telomerase activity in fish muscle by biological and environmental factors

Telomerase expression has long been linked to promotion of tumor growth and cell proliferation in mammals. Interestingly, telomerase activity (TA) has been detected in skeletal muscle for a variety of fish species. Despite this being a unique feature in fish, very few studies have investigated the potential role of TA in muscle. The present study was set to prove the concepts that muscle telomerase in fish is related to body growth, and more specifically, to muscle cell proliferation and apoptosis in vivo. Moreover, muscle TA can be influenced by biotic factors and modulated by environmental stress. Using three fish species, mangrove red snapper (Lutjanus argentimaculatus), orange-spotted grouper (Epinephelus coioides), and marine medaka (Oryzias melastigma), the present work reports for the first time that fish muscle TA was sensitive to the environmental stresses of starvation, foodborne exposure to benzo[a]pyrene, and hypoxia. In marine medaka, muscle TA was coupled with fish growth during early life stages. Upon sexual maturation, muscle TA was confounded by sex (female>male). Muscle TA was significantly correlated with telomerase reverse transcriptase (TERT) protein expression (Pearson correlation r=0.892; p≤0.05), which was coupled with proliferating cell nuclear antigen (PCNA) cell proliferation, but not associated with apoptosis (omBax/omBcl2 ratio) in muscle tissue. The results reported here have bridged the knowledge gap between the existence and function of telomerase in fish muscle. The underlying regulatory mechanisms of muscle TA in fish warrant further exploration for comparison with telomerase regulation in mammals.

Insight into the transgenerational effect of benzo[a]pyrene on bone formation in a teleost fish (Oryzias latipes) ☆

Recent cross-generational studies in teleost fish have raised the awareness that high levels of benzo[a]pyrene (BaP) could affect skeletal integrity in the directly exposed F0 and their F1-F2. However, no further details were provided about the causes for abnormalities on the molecular and cellular level and the persistence of such sub-organismal impairments at the transgenerational scale (beyond F2). Adult Oryzias latipes were exposed to 1μg/L BaP for 21days. The F1-F3 were examined for skeletal deformities, histopathological alterations of vertebral bodies and differential expression of key genes of bone metabolism. Significant increase of dorsal-ventral vertebral compression was evident in ancestrally exposed larvae. Histopathological analysis revealed abnormal loss of notochord sheath, a lack of notochord epithelial integrity, reduced bone tissue and decreased osteoblast abundance. A significant downregulation of ATF4 and/or osterix and a high biological variability of COL10, coupled with a significant deregulation of SOX9a/b in the F1-F3 suggest that ancestral BaP exposure most likely perturbed chordoblasts, chondroblast and osteoblast differentiation, resulting in defective notochord sheath repair and rendering the vertebral column more vulnerable to compression. The present findings provide novel molecular and cellular insights into BaP-induced transgenerational bone impairment in the unexposed F3. 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 basic 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 the genesis of skeletal diseases in humans.

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.

Sex-dependent telomere shortening, telomerase activity and oxidative damage in marine medaka Oryzias melastigma during aging

Marine medaka Oryzias melastigma at 4months (young), 8months (middle-aged) and 12months old (senior) were employed to determine age-associated change of sex ratios, sex hormones, telomere length (TL), telomerase activity (TA), telomerase transcription (omTERT) and oxidative damage in the liver. Overall, O. melastigma exhibited gradual senescence, sex differences in longevity (F>M), TL (F>M) and oxidative damage (F5kb), TA and omTERT expression (p≤0.01), and negatively correlated with liver DNA oxidation (p≤0.05). The results suggest high levels of E2 in female O. melastigma may retard TL shortening by enhancing TA via TERT transcription and/or reducing oxidative DNA damage. The findings support TL shortening as a biomarker of aging and further development of accelerated TL shortening, abnormal suppression of TA and excessive oxidative DNA damage as early molecular endpoints, indicative of advanced/premature aging in marine medaka/fish.

Identification of Molecular Targets for 4,5-Dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) in Teleosts: New Insight into Mechanism of Toxicity

Environmental pollutants are capable of concomitantly inducing diverse toxic effects. However, it is largely unknown which effects are directly induced and which effects are secondary, thus calling for definitive identification of the initiating molecular event for a pollutant to elucidate the mechanism of toxicity. In the present study, affinity pull-down assays were used to identify target proteins for 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT), a costal pollutant of emerging concern, in various tissues (e.g., brain, liver, plasma, and gonad) from marine medaka (Oryzias melastigma) and zebrafish (Danio rerio). Pull-down results showed that, in male and female brains from medaka and zebrafish, DCOIT had a consistently high affinity for G protein alpha subunits (Gα), suggesting the targeted effects of DCOIT on signaling transduction from G protein-coupled receptors (GPCRs) and an extrapolatable mode of action in teleost brains. Validation using recombinant proteins and molecular docking analysis confirmed that binding of DCOIT to Gα protein competitively inhibited its activation by substrate. Considering the involvement of GPCRs in the regulation of myriad biological processes, including the hypothalamus-pituitary-gonadal-liver axis, binding of DCOIT to upstream Gα proteins in the brain may provide a plausible explanation for the diversity of toxic effects resulting from DCOIT challenge, especially abnormal hormonal production through the mitogen-activated protein kinase pathway. A new mechanism of action based on GPCR signaling is thus hypothesized for endocrine disrupting chemicals and warrants further research to clearly elucidate the link between GPCR signaling and endocrine disruption.

Modification of the plasma complement protein profile by exogenous estrogens is indicative of a compromised immune competence in marine medaka (Oryzias melastigma)

ABSTRACT Growing evidence suggests that the immune system of teleost is vulnerable to xenoestrogens, which are ubiquitous in the marine environment. This study detected and identified the major circulatory immune proteins deregulated by 17&agr;‐ethinylestradiol (EE2), which may be linked to fish susceptibility to pathogens in the marine medaka, Oryzias melastigma. Fish immune competence was determined using a host resistance assay to pathogenic bacteria Edwardsiella tarda. Females were consistently more susceptible to infection‐induced mortality than males. Exposure to EE2 could narrow the sex gap of mortality by increasing infection‐induced death in male fish. Proteomic analysis revealed that the major plasma immune proteins of adult fish were highly sexually dimorphic. EE2 induced pronounced sex‐specific changes in the plasma proteome, with the male plasma composition clearly becoming “feminised”. Male plasma was found to contain a higher level of fibrinogens, WAP63 and ependymin‐2‐like protein, which are involved in coagulation, inflammation and regeneration. For the first time, we demonstrated that expression of C1q subunit B (C1Q), an initiating factor of the classical complement pathway, was higher in males and was suppressed in both sexes in response to EE2 and bacterial challenge. Moreover, cleavage and post‐translational modification of C3, the central component of the complement system, could be altered by EE2 treatment in males (C3dg down; C3g up). Multiple regression analysis indicated that C1Q is possibly an indicator of fish survival, which warrants further confirmation. The findings support the potential application of plasma immune proteins for prognosis/diagnosis of fish immune competence. Moreover, this study provides the first biochemical basis of the sex‐differences in fish immunity and how these differences might be modified by xenoestrogens. HighlightsEE2 exposure reduces fish resistance to pathogen infection.The plasma immune proteins of adult fish are sexually dimorphic.Exogenous EE2 can modify the immune plasma proteome.The interplay between plasma C1Q and exogenous EE2 warrants further investigation.

The development of cellular immune defence in marine medaka Oryzias melastigma

Environmentally induced alterations of the immune system during sensitive developmental stages may manifest as abnormalities in immune organ configuration and/or immune cell differentiation. These not only render the early life stages more vulnerable to pathogens, but may also affect the adult immune competence. Knowledge of these sensitive periods in fish would provide an important prognostic/diagnostic tool for aquatic risk assessment of immunotoxicants. The marine medaka Oryzias melastigma is an emerging seawater fish model for immunotoxicology. Here, the presence and onset of four potentially sensitive periods during the development of innate and adaptive cellular immune defence were revealed in O. melastigma: 1.) initiation of phagocyte differentiation, 2.) migration and expansion of lymphoid progenitor cells, 3.) colonization of immune organs through lymphocyte progenitors and 4.) establishment of immune competence in the thymus. By using an established bacterial resistance assay for O. melastigma, larval immune competence (from newly hatched 1dph to 14dph) was found concomitantly increased with advanced thymus development and the presence of mature T-lymphocytes. A comparison between the marine O. melastigma and the freshwater counterpart Oryzias latipes disclosed a disparity in the T-lymphocyte maturation pattern, resulting in differences in the length of T-lymphocyte maturation. The results shed light on a potential difference between seawater and freshwater medaka in their sensitivity to environmental immunotoxicants. Further, medaka immune system development was compared and contrasted to economically important fish. The present study has provided a strong scientific basis for advanced investigation of critical windows for immune system development in fish.

Sex-specific immunomodulatory action of the environmentalestrogen 17α-ethynylestradiol alongside with reproductive impairment in fish

Estrogenic endocrine disrupting chemicals (EEDCs) are present ubiquitously in sediments and aquatic ecosystems worldwide. The detrimental impact of EEDCs on the reproduction of wildlife is widely recognized. Increasing evidence shows the immunosuppressive effects of EEDCs in vertebrates. Yet, no studies have considered concomitantly EEDC-induced impacts on reproductive impairment and immune suppression in vivo, which are deemed essential for risk assessment and environmental monitoring. In this study, EE2 was used as a representative EEDC, for parallel evaluation of EEDC-induced immune suppression (immune marker gene expression, leukocyte numbers, host resistance assay, and immune competence index) and reproductive impairment (estrogen responsive gene expression, fecundity, fertilization success, hatching success, and reproductive competence index) in an established fish model (marine medaka Oryzias melastigma), considering sex-specific induction and adaptation and recovery responses under different EE2 exposure scenarios. The findings in marine medaka reveal distinct sex differences in the EE2-mediated biological responses. For female fish, low concentration of exogenous EE2 (33 ng/L) could induce hormesis (immune enhancement), enable adaptation (restored reproduction) and even boost fish resistance to bacterial challenge after abatement of EE2. However, a prolonged exposure to high levels of EE2 (113 ng/L) not only impaired F0 immune function, but also perturbed females recovering from reproductive impairment, resulting in a persistent impact on the F1 generation output. Thus, for female fish, the exposure concentration of EE2 is more critical than the dose of EE2 in determining the impacts of EE2 on immune function and reproduction. Conversely, male fish are far more sensitive than females to the presence of low levels of exogenous EE2 in water and the EE2-mediated biological impacts are clearly dose-dependent. It is also evident in male fish that direct contact of EE2 is essential to sustain impairments of immune competence and reproductive output as well as deregulation of immune function genes in vivo. The immunomodulatory pathways altered by EE2 were deciphered for male and female fish, separately. Downregulation of hepatic tlr3 and c3 (in female) and tlr3, tlr5 and c3 (in male) may be indicative of impaired fish immune competence. Taken together, impaired immune competence in the EE2-exposed fish poses an immediate thread on the survival of F0 population. Impaired reproduction in the EE2-exposed fish can directly affect F1 output. Parallel evaluation of immune competence and reproduction are important considerations when assessing the risk of sublethal levels of EE2/EEDCs in aquatic environments.