From metabolism to behaviour - Multilevel effects of environmental methamphetamine concentrations on fish.

Methamphetamine (METH) is a concerning drug of abuse that produces strong psychostimulant effects. The use of this substance, along with the insufficient removal in the sewage treatment plants, leads to its occurrence in the environment at low concentrations. In this study, brown trout (Salmo trutta fario) were exposed to 1 µg/L of METH as environmental relevant concentration for 28 days in order to elucidate the complex effects resulting from the drug, including behaviour, energetics, brain and gonad histology, brain metabolomics, and their relations. Trout exposed to METH displayed lowered activity as well as metabolic rate (MR), an altered morphology of brain and gonads as well as changes in brain metabolome when compared to controls. Increased activity and MR were correlated to an increased incidence of histopathology in gonads (females - vascular fluid and gonad staging; males - apoptotic spermatozoa and peritubular cells) in exposed trout compared to controls. Higher amounts of melatonin in brain were detected in exposed fish compared to controls. Tyrosine hydroxylase expression in locus coeruleus was related to the MR in exposed fish, but not in the control. Brain metabolomics indicated significant differences in 115 brain signals between control and METH exposed individuals, described by the coordinates within the principal component analyses (PCA) axes. These coordinates were subsequently used as indicators of a direct link between brain metabolomics, physiology, and behaviour - as activity and MR varied according to their values. Exposed fish showed an increased MR correlated with the metabolite position in PC1 axes, whereas the control had proportionately lower MR and PC1 coordinates. Our findings emphasize the possible complex disturbances in aquatic fauna on multiple interconnected levels (metabolism, physiology, behaviour) as a result of the presence of METH in aquatic environments. Thus, these outcomes can be useful in the development of AOP's (Adverse Outcome Pathways).

Effects of the synthetic progestin levonorgestrel on some aspects of thyroid physiology in common carp (Cyprinus carpio).

The objective of the present study was to assess the effects of levonorgestrel (LNG), a synthetic progestin, on early development and the thyroid system of carp using morphological, histological, immunohistochemical, and gene expression analysis. Fish were exposed to LNG at three levels (3, 31, and 310 ng L-1) from eggs to the onset of juvenile stage (47 days). LNG had no significant effect on early development in common carp or on the occurrence of morphological anomalies. No pathological alterations of the thyroid follicles were found. Immunohistochemical examination of the thyroid follicles using antibodies against thyroxin did not show any differences in fish exposed to 310 ng L-1 LNG compared to the controls. mRNA expression of iodothyronine deiodinases (dio1, 2, 3) was differentially affected by LNG treatment during carp development. Most importantly, dio3 was markedly downregulated in fish exposed to all three LNG levels compared to the controls at the conclusion of the experiment (47 days post-fertilization). A decrease in dio1 or dio3 or an increase in dio2 transcription observed at different time points of the study may be a sign of hypothyroidism. mRNA expression of genes npr, esr1, and esr2b in the body and npr and esr2b in the head of fish exposed to 310 ng L-1 LNG was significantly upregulated compared to the solvent control group at the end of the test. Together, these results show that levonorgestrel caused parallel changes in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-gonad axes.

Immunohistochemical investigation of epithelial, mesenchymal, neuroectodermal, immune and endocrine markers in sterlet (Acipenser ruthenus), shortnose sturgeon (Acipenser brevirostrum) and common carp (Cyprinus carpio).

Immunohistochemistry (IHC) is a laboratory method widely used to characterize tissue and cell origin, both in human and veterinary medicine. In fish, however, little is known about staining characteristics of most tissue types, and especially for less studied chondrostean fish. The aim of this study was to examine the specificity of various immunohistochemical markers in tissues of chondrostean and teleostean fish and to validate diagnostic tests. Sterlet (Acipenser ruthenus L.), shortnose sturgeon (Acipenser brevirostrum) and common carp (Cyprinus carpio L.) were examined. Markers were chosen as representatives of epithelial (cytokeratin AE1/AE3), mesenchymal (vimentin), neuroectodermal (S-100 protein), lymphoid (leukocyte common antigen, LCA) and endocrine (thyroglobulin, thyroxin) tissues and organs. Applied antibodies were of monoclonal or polyclonal mammalian origin and primarily intended for human medicine research or diagnostic application. No species differences were obvious while examining sterlet, shortnose sturgeon and carp. Cytokeratin AE1/AE3, vimentin, S-100 protein and thyroxin were positive on targeted tissues and structures. Leukocyte common antigen (LCA) and thyroglobulin were negative on targeted structures, however, and with clear cross-reactivity on non-targeted tissues (vascular wall, granulocytes). Conclusive results were obtained when using polyclonal antibodies with dilution adjusted to laboratory practice, while application of ready-to-use (RTU) kits with pre-diluted antibodies or monoclonal antibodies often showed conflicting or inconclusive results.

Environmental concentration of methamphetamine induces pathological changes in brown trout (Salmo trutta fario).

Methamphetamine, mainly consumed as an illicit drug, is a potent addictive psychostimulant that has been detected in surface water at concentrations ranging from nanograms to micrograms per litre, especially in Middle and East Europe. The aim of this study was to expose brown trout (Salmo trutta fario) to environmental (1 µg L-1) and higher (50 µg L-1) concentrations of methamphetamine for 35 days with a four-day depuration phase to assess the possible negative effects on fish health. Degenerative liver and heart alterations, similar to those described in mammals, were observed at both concentrations, although at different intensities. Apoptotic changes in hepatocytes, revealed by activated caspase-3, were found in exposed fish. The parent compound and a metabolite (amphetamine) were detected in fish tissues in both concentration groups, in the order of kidney > liver > brain > muscle > plasma. Bioconcentration factors ranged from 0.13 to 80. A therapeutic plasma concentration was reached for both compounds in the high-concentration treatment. This study indicates that chronic environmental concentrations of methamphetamine can lead to health issues in aquatic organisms.

Mature dendritic cells correlate with favorable immune infiltrate and improved prognosis in ovarian carcinoma patients.

A high density of tumor-infiltrating CD8+ T cells and CD20+ B cells correlates with prolonged survival in patients with a wide variety of human cancers, including high-grade serous ovarian carcinoma (HGSC). However, the potential impact of mature dendritic cells (DCs) in shaping the immune contexture of HGSC, their role in the establishment of T cell-dependent antitumor immunity, and their potential prognostic value for HGSC patients remain unclear. We harnessed immunohistochemical tests and biomolecular analyses to demonstrate that a high density of tumor-infiltrating DC-LAMP+ DCs is robustly associated with an immune contexture characterized by TH1 polarization and cytotoxic activity. We showed that both mature DCs and CD20+ B cells play a critical role in the generation of a clinically-favorable cytotoxic immune response in HGSC microenvironment. In line with this notion, robust tumor infiltration by both DC-LAMP+ DCs and CD20+ B cells was associated with most favorable overall survival in two independent cohorts of chemotherapy-naïve HGSC patients. Our findings suggest that the presence of mature, DC-LAMP+ DCs in the tumor microenvironment may represent a novel, powerful prognostic biomarker for HGSC patients that reflects the activation of clinically-relevant anticancer immunity.

Markers of HPV infection and survival in patients with head and neck tumors.

The purpose of this study was to determine whether changes in human papillomavirus (HPV) DNA prevalence in oral rinses and/or HPV-specific antibody levels in the sera of patients with oral/oropharyngeal cancer have prognostic significance. One hundred and forty-two patients with oral/oropharyngeal tumors were enrolled. The presence of HPV DNA was assayed in tumor tissue and oral rinses and HPV-specific antibodies were assessed in the sera. Oral rinses were collected before treatment and one year after the treatment. Sera were drawn before treatment, one month, and one year after the end of the treatment. Altogether, 59.2% of tumors were HPV positive. The presence of HPV DNA in the tumors correlated with HPV DNA positivity in oral rinses and with HPV-specific antibodies in the sera. Out of 66 patients with HPV-positive oral rinses at enrolment, 84.8% became negative at one-year follow-up, while most patients remained seropositive for HPV-specific antigens. However, the mean titers of HPV16 E6 and/or E7 antibodies at follow-up were significantly lower. Of 16 patients with recurrences at follow-up (alive on second sampling), six were positive at enrolment for HPV16 E6 and/or E7 antibodies. In five of these, no decrease in antibody levels was observed. Titers of antibodies specific for HPV16 capsid antigens did not change during the follow-up. Our data suggest that the detection of antibodies specific for the HPV 16 E6 and E7 oncoproteins may serve not only as a marker of HPV etiology, but also as a marker of recurrence and a prognostic indicator in patients with HPV-positive tumors.