E6/E7 mRNA Expression of the Most Prevalent High-Risk HPV Genotypes in Cervical Samples from Serbian Women.

Cervical cancer caused by persistent infection with HR HPV genotypes is the second leading cause of death in women aged 15 to 44 in Serbia. The expression of the E6 and E7 HPV oncogenes is considered as a promising biomarker in diagnosing high-grade squamous intraepithelial lesions (HSIL). This study aimed to evaluate HPV mRNA and DNA tests, compare the results according to the severity of the lesions, and assess the predictive potential for the diagnosis of HSIL. Cervical specimens were obtained at the Department of Gynecology, Community Health Centre Novi Sad, Serbia, and the Oncology Institute of Vojvodina, Serbia, during 2017-2021. The 365 samples were collected using the ThinPrep Pap test. The cytology slides were evaluated according to the Bethesda 2014 System. Using a real-time PCR test, HPV DNA was detected and genotyped, while the RT-PCR proved the presence of E6 and E7 mRNA. The most common genotypes in Serbian women are HPV 16, 31, 33, and 51. Oncogenic activity was demonstrated in 67% of HPV-positive women. A comparison of the HPV DNA and mRNA tests to assess the progression of cervical intraepithelial lesions indicated that higher specificity (89.1%) and positive predictive value (69.8-78.7%) were expressed by the E6/E7 mRNA test, while higher sensitivity was recorded when using the HPV DNA test (67.6-88%). The results determine the higher probability of detecting HPV infection by 7% provided by the mRNA test. The detected E6/E7 mRNA HR HPVs have a predictive potential in assessing the diagnosis of HSIL. The oncogenic activity of HPV 16 and age were the risk factors with the strongest predictive values for the development of HSIL.

Characterization of the ERK1/2 phosphorylation profile in human and fish liver cells upon exposure to chemicals of environmental concern.

We developed phospho-ERK1/2 ELISA for human and rainbow trout liver cells, employing HepG2 and RTL-W1 cell lines as models. The assay was applied to detect changes in ERK1/2 activity for nine chemicals, added over a wide concentration range and time points. Cell viability was measured to separate ERK1/2 regulation from cytotoxicity. Perfluorooctane sulfonate and carbendazim did not change ERK1/2 activity; influence on ERK1/2 due to cytotoxicity was indicated for tributyltin and cypermethrin. Mancozeb, benzo[a]pyrene, and bisphenol A stimulated ERK1/2 up to ∼2- (HepG2) and 1.5 (RTL-W1)-fold, though the kinetics differed between chemicals and cell lines. Bisphenol A and benzo[a]pyrene were the most potent concentration-wise, altering ERK1/2 activity in pM (HepG2) to nM (RTL-W1) range. While atrazine and ibuprofen increased ERK1/2 activity by ∼2-fold in HepG2, they did not initiate an appreciable response in RTL-W1. This assay proved to be a sensitive, medium- to high-throughput tool for detecting unrecognized ERK1/2-disrupting chemicals.

Interaction of organotin compounds with three major glutathione S-transferases in zebrafish.

Glutathione S-transferases (GSTs) play an important role in cellular detoxification as enzymatic mediators of glutathione (GSH) conjugation with a wide range of deleterious compounds, enabling their easier extrusion out of the organism. GSTs are shown to interact with organotin compounds (OTCs), known environmental pollutants, either as substrates, serving as electrophilic targets to the nucleophilic attack of GSH, or as noncompetitive inhibitors by binding to GST active sites and disrupting their enzymatic functions. There is a wide range of deleterious biological effects caused by OTCs in low concentration range. Their environmental concentrations, further potentiated by bioaccumulation in aquatic organisms, correspond with inhibitory constants reported for Gsts in zebrafish, which implies their environmental significance. Therefore, our main goal in this study was to analyze interactions of three major zebrafish Gsts - Gstp1, Gstr1, and Gstt1a - with a series of ten environmentally relevant organotin compounds. Using previously developed Gst inhibition assay with recombinant Gst proteins and fluorescent monochlorobimane as a model substrate, we determined Gst inhibitory constants for all tested OCTs. Furthermore, in order to elucidate nature of Gst interactions with OTCs, we determined type of interactions between tested Gsts and the strongest OTC inhibitors. Our results showed that OTCs can interact with zebrafish Gsts as competitive, noncompetitive, or mixed-type inhibitors. Determined types of interactions were additionally confirmed in silico by molecular docking studies of tested OTCs with newly developed Gst models. In silico models were further used to reveal structures of tested Gsts in more detail and identify crucial amino acid residues which interact with OTCs within Gst active sites. Our results revealed more extensive involvement of Gstr1 and Gstp1 in detoxification of numerous tested OTCs, with low inhibitory constants in nanomolar to low micromolar range and different types of inhibition, whereas Gstt1a noncompetitively interacted with only two tested OTCs with significantly higher inhibitory constants.

Molecular characterization of zebrafish Gstr1, the only member of teleost-specific glutathione S- transferase class.

Glutathione S-transferases (GSTs) are multifunctional phase II detoxification enzymes with primary function of glutathione conjugation of various endogenous and exogenous compounds. Teleost-specific Gstr1 in zebrafish (Danio rerio) was previously shown to have high expression in toxicologically relevant tissues and high activity towards model substrates. The aim of this study was a detailed functional characterization of zebrafish Gstr1. Molecular docking analyses were used to get novel insight into structural characteristics of Gstr1 and elucidation of the mechanistic interactions with both GSH and various Gstr1 substrates or inhibitors. An initial screening inhibition assay performed using model fluorescence substrate monochlorobimane (MCB) revealed interactions of different endogenous compounds and environmentally relevant xenobiotics with zebrafish Gstr1. All interacting compounds were further analyzed to determine their inhibition type and Ki values. Our data revealed that pregnenolone, progesterone, testosterone, DHEAS and corticosterone competitively inhibited transformation of MCB by Gstr1 with the calculated Ki values in the range 14-26 µM, implying that these hormones are physiological substrates of zebrafish Gstr1. Estrogens had no effect on Gstr1 activity. Taurochenodeoxycholate (TCDC) expressed lower inhibition potency toward Gstr1 with the Ki value of 33 µM. Among tested xenobiotics tributyltin chloride and rifampicin non-enzymatically bound Gstr1 enzyme (the calculated Ki values are 0.26 µM and 65 µM, respectively) and inhibited its activity, showing that these compounds are reversible noncompetitive inhibitors of zebrafish Gstr1. Insecticide diazinon competitively inhibited Gstr1 activity with calculated Ki value of 27 µM, while other Gstr1-interacting insecticides, chlorpyrifos-methyl (CPF-methyl) and malathion, showed allosteric activation-like effect. Among tested pharmaceuticals, tetracycline, erythromycin and methotrexate demonstrated competitive type of inhibition with the calculated Ki values of 17.5, 36.5 and 29 µM, respectively. In summary, we suggest that zebrafish Gstr1 has an important role in steroidogenesis, metabolism and/or physiological actions of androgens, but not estrogens in fish. Finally, our results imply the role of Gstr1 in metabolism of xenobiotics and protection of fish against deleterious environmental contaminants such as organophosphate insecticides and pharmaceuticals.