Interplay between Rac1b and Sodium Iodide symporter expression in thyroid and breast cancers

Rac1b, an alternative isoform of the small GTPase RAC1, has recently be shown to be present in thyroid tissue and overexpressed in thyroid cancer cells, particularly in a subset of papillary thyroid carcinomas carrying the activating mutation BRAFV600E that are associated with an unfavorable outcome. On the other hand, RAC1 seems to be involved in the upregulation of NIS, the glycoprotein responsible for iodide uptake that allows the use of 131 I as a diagnostic and therapeutic tool, in thyroid cancer. However, NIS expression levels and iodine uptake in thyroid cancer cells are reduced when compared to normal tissue. Also, B-Raf V600E mutation has been shown to correlate with a lower expression of NIS. RAC1b overexpression has also been documented in breast cancer. This hyperactivatable variant was shown to be able to compete with and inhibit RAC1 endogenous activity in several signaling pathways. Breast carcinomas also express NIS but at levels too low to warrant treatment with 131I. Thus, in order to understand the regulatory mechanisms of NIS expression we aimed to evaluate the balance of RAC1/1b effect in NIS mRNA expression in follicular cell derived thyroid tumor samples, as well as, in a cell line derived from normal thyroid and in breast cancer cell lines. Understanding the necessary switch to increase NIS expression in cancer cells, would open a new window of opportunity to fight thyroid tumor resistance to radioiodine therapy and develop and possible treatment by the radiodide uptake therapy in breast cancer in a selective way

Application of new approach methodologies in molecular toxicology:A case study on metabolism and cellular stress responses from glutathione conjugation products of trichloroethylene

Traditional safety assessments often rely on animal models, which may not reflect human responses to chemical exposures and raise ethical concerns. Despite the increasing efforts to develop new approach methodologies (NAMs), their integration into regulatory decision-making processes is still challenging. The European Union has played a key role in the advancement of NAMs through funding projects like the EU-ToxRisk project. This PhD thesis, funded by EU-ToxRisk, focuses on the multi-organ metabolism of chemicals like trichloroethylene (TCE), a halogenated alkene which has been extensively associated with toxicity and carcinogenicity. TCE’s nephrotoxic effects result from multi-organ metabolism and bioactivation via the mercapturic acid pathway. Based on its chemical structure, substitution of chlorine-atoms of TCE by glutathione (GSH) theoretically can lead to the formation of three regioisomeric GSH-conjugates: S-(1,2-trans-dichlorovinyl)-glutathione (1,2-trans-DCVG), S-(1,2-cis-dichlorovinyl)-glutathione (1,2-cis-DCVG) and S-(2,2-dichlorovinyl)-glutathione (2,2-DCVG). However, at the time of the start of this PhD project, there was only experimental evidence for two regioisomers, namely 1,2-trans-DCVG and 2,2-DCVG. In chapter 2, a novel LC-MS method was developed to separate and quantify DCVG regioisomers and their corresponding cysteine S-conjugate (DCVCs) and N-acetyl-L-cysteine-S-conjugates (DCV-NACs). This method revealed significant differences in the formation profile of these regioisomers when comparing incubations in rat and human liver fractions. Novel methods were also developed for the synthesis and purification of all three regioisomers of DCVG, DCVC, and DCV-NAC. Using a β-lyase-mimetic model and 4-(p-nitrobenzyl)pyridine (NBP) as a model nucleophile, we demonstrated that only 1,2-trans-DCVC and 1,2-cis-DCVC formed NBP-reactive products, suggesting their higher reactivity. The purified regioisomers of DCVG and DCVC were used to investigate their cellular effects in different cellular models. In chapter 3, six different human in vitro systems representing target organs of TCE were used to compare altered transcriptional responses upon exposure to the major conjugates formed by human liver fractions, 1,2-trans-DCVG/C and 2,2-DCVG/C (chapter 2). Viability studies showed that 1,2-DCVC affected the viability of all six cell models in a concentration-dependent manner, while 1,2-DCVG only affected the kidney model (RPTEC/TERT1). The kidney and liver models showed the strongest transcriptomic responses to both 1,2-DCVG and 1,2-DCVC. Transcripts associated with Nrf2-mediated oxidative stress response and ATF4-mediated unfolded protein response (UPR) were upregulated in all cell types at high concentrations of 1,2-DCVC exposure. In chapter 4, the renal cellular effects of 1,2-cis-DCVC and 1,2-cis-DCVG were investigated, showing significant differences in their effects on cell viability and mitochondrial respiration compared to 1,2-trans-DCVG/C and 2,2-DCVG/C isomers. Despite similar reactivities of the β-lyase products of 1,2-trans-DCVC and 1,2-cis-DCVC, 1,2-cis-DCVC did not affect cell viability of RPTEC/TERT1 cells at any of the concentrations tested. The rates of metabolism of the three DCVG regioisomers by RPTEC/TERT1 cells were investigated. All three DCVGs were degraded rapidly at comparable rates, with simultaneous formation of their corresponding cysteinylglycinyl-S-conjugates (DCV-CysGly). Three metabolites corresponding to N-γ-glutamyl-S-(dichlorovinyl)-L-cysteine (DCV-CysGlu) conjugates were identified, marking the first time this pathway is reported in TCE metabolism. In chapter 5, cellular responses were determined after repeated daily exposure for 7 days to non-toxic concentrations of TCE-conjugates. Both 1,2-trans-DCVC and 1,2-trans-DCVG started to cause a decrease in cell number after 5 days of exposure, with 1,2-trans-DCVC being slightly more toxic. 2,2-DCVG, the major DCVG-regioisomer formed by human liver fractions (chapter 2), only produced minor Nrf2-responses when exposed for 24 h at high concentration (>250 µM) to RPTEC/TERT1-cells. This confirms that this regioisomer seems to be of low toxicological concern for humans. In conclusion, by integrating kinetic and dynamic techniques for the evaluation of compounds that go through the GSH conjugation pathway, the work done in this PhD thesis offers confidence in the successful application of NAMs to support chemical risk assessment

The economic costs of biological invasions in Central and South America: a first regional assessment

Invasive alien species are responsible for a high economic impact on many sectors worldwide. Nevertheless, there is a scarcity of studies assessing these impacts in Central and South America. Investigating costs of invasions is important to motivate and guide policy responses by increasing stakeholders’ awareness and identifying action priorities. Here, we used the InvaCost database to investigate (i) the geographical pattern of biological invasion costs across the region; (ii) the monetary expenditure across taxa and impacted sectors; and (iii) the taxa responsible for more than 50% of the costs (hyper-costly taxa) per impacted sector and type of costs. The total of reliable and observed costs reported for biological invasions in Central and South America was USD 102.5 billion between 1975 and 2020, but about 90% of the total costs were reported for only three countries (Brazil, Argentina and Colombia). Costs per species were associated with geographical regions (i.e., South America, Central America and Islands) and with the area of the countries in km2. Most of the expenses were associated with damage costs (97.8%), whereas multiple sectors (77.4%), agriculture (15%) and public and social welfare (4.2%) were the most impacted sectors. Aedes spp. was the hyper-costly taxon for the terrestrial environment (costs of USD 25 billion) and water hyacinth (Eichhornia crassipes) was the hyper-costly taxon for the aquatic environment (USD 179.9 million). Six taxa were classified as hyper-costly for at least one impacted sector and two taxa for at least one type of cost. In conclusion, invasive alien species caused billions of dollars of economic burden in Central and South America, mainly in large countries of South America. Costs caused by invasive alien species were unevenly distributed across countries, impacted sectors, types of costs and taxa (hyper-costly taxa). These results suggest that impacted sectors should drive efforts to manage the species that are draining financial sources.info:eu-repo/semantics/publishedVersio

Development of a high-throughput in vitro screening method for the assessment of cell-damaging activities of snake venoms

Snakebite envenoming is a globally important public health issue that has devastating consequences on human health and well-being, with annual mortality rates between 81,000 and 138,000. Snake venoms may cause different pathological effects by altering normal physiological processes such as nervous transfer and blood coagulation. In addition, snake venoms can cause severe (local) tissue damage that may result in life-long morbidities, with current estimates pointing towards an additional 450,000 individuals that suffer from permanent disabilities such as amputations, contractions and blindness. Despite such high morbidity rates, research to date has been mainly focusing on neurotoxic and haemotoxic effects of snake venoms and considerably less on venom-induced tissue damage. The molecular mechanisms underlaying this pathology include membrane disruption and extracellular matrix degradation. This research describes methods used to study the (molecular) mechanisms underlaying venom-induced cell- and tissue damage. A selection of cellular bioassays and fluorescent microscopy were used to study cell-damaging activities of snake venoms in multi-well plates, using both crude and fractionated venoms. A panel of 10 representative medically relevant snake species was used, which cover a large part of the geographical regions most heavily affected by snakebite. The study comprises both morphological data as well as quantitative data on cell metabolism and viability, which were measured over time. Based on this data, a distinction could be made in the ways by which viper and elapid venoms exert their effects on cells. We further made an effort to characterise the bioactive compounds causing these effects, using a combination of liquid chromatography methods followed by bioassaying and protein identification using proteomics. The outcomes of this study might prove valuable for better understanding venom-induced cell- and tissue-damaging pathologies and could be used in the process of developing and improving snakebite treatments

Non-English languages enrich scientific knowledge : The example of economic costs of biological invasions

We contend that the exclusive focus on the English language in scientific researchmight hinder effective communication between scientists and practitioners or policymakerswhose mother tongue is non-English. This barrier in scientific knowledge and data transfer likely leads to significant knowledge gaps and may create biases when providing global patterns in many fields of science. To demonstrate this, we compiled data on the global economic costs of invasive alien species reported in 15 non-English languages. We compared it with equivalent data from English documents (i.e., the InvaCost database, the most up-to-date repository of invasion costs globally). The comparison of both databases (similar to 7500 entries in total) revealed that non-English sources: (i) capture a greater amount of data than English sources alone (2500 vs. 2396 cost entries respectively); (ii) add 249 invasive species and 15 countries to those reported by English literature, and (iii) increase the global cost estimate of invasions by 16.6% (i.e., US$ 214 billion added to 1.288 trillion estimated fromthe English database). Additionally, 2712 cost entries - not directly comparable to the English database - were directly obtained frompractitioners, revealing the value of communication between scientists and practitioners. Moreover, we demonstrated how gaps caused by overlooking non-English data resulted in significant biases in the distribution of costs across space, taxonomic groups, types of cost, and impacted sectors. Specifically, costs from Europe, at the local scale, and particularly pertaining to management, were largely under-represented in the English database. Thus, combining scientific data from English and non-English sources proves fundamental and enhances data completeness. Considering non-English sources helps alleviate biases in understanding invasion costs at a global scale. Finally, it also holds strong potential for improving management performance, coordination among experts (scientists and practitioners), and collaborative actions across countries. Note: non-English versions of the abstract and figures are provided in Appendix S5 in 12 languages. (c) 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/ by/4.0/).Peer reviewe

Corrigendum to “Bioactivation of trichloroethylene to three regioisomeric glutathione conjugates by liver fractions and recombinant human glutathione transferases: Species differences and implications for human risk assessment” [Toxicol. Lett. 341 (2021) 94–106]

The authors regret that in Fig. 1 the letters indicating the enzymes involved in the reactions were not added to the arrows. A corrected Figure is shown below. [Figure presented] Fig. 1. Regioselective GSH-conjugation of trichloroethylene (TCE) and subsequent processing to corresponding mercapturic acids (N-acetylcysteine S-conjugates) and beta-lyase-dependent bioactivation of cysteine S-conjugates to reactive products. Enzymes involved: a. glutathione S-transferases; b. gamma-glutamyltransferase; c. cysteinyl-glycine dipeptidase; d. cysteine conjugate beta-lyase; e. cysteine conjugate N-acetyltransferase; f. aminoacylase. Formation of sulfoxides of mercapturic acids is not shown to avoid an overcomplicated figure. The authors would like to apologise for any inconvenience caused

Bioactivation of trichloroethylene to three regioisomeric glutathione conjugates by liver fractions and recombinant human glutathione transferases:Species differences and implications for human risk assessment

Enzymatic conjugation of glutathione (GSH) to trichloroethylene (TCE) followed by catabolism to the corresponding cysteine-conjugate, S-(dichlorovinyl)-L-cysteine (DCVC), and subsequent bioactivation by renal cysteine conjugate beta-lyases is considered to play an important role in the nephrotoxic effects observed in TCE-exposed rat and human. In this study, it is shown for the first time that three regioisomers of GSH-conjugates of TCE are formed by rat and human liver fractions, namely S-(1,2-trans-dichlorovinyl)-glutathione (1,2-trans-DCVG), S-(1,2-cis-dichlorovinyl)-glutathione (1,2-cis-DCVG) and S-(2,2-dichlorovinyl)-glutathione (2,2-DCVG). In incubations of TCE with rat liver fractions their amounts decreased in order of 1,2-cis-DCVG > 1,2-trans-DCVG > 2,2-DCVG. Human liver cytosol showed a more than 10-fold lower activity of GSH-conjugation, with amounts of regioisomers decreasing in order 2,2-DCVG > 1,2-trans-DCVG > 1,2-cis-DCVG. Incubations with recombinant human GSTs suggest that GSTA1-1 and GSTA2-2 play the most important role in human liver cytosol. GSTP1-1, which produces regioisomers in order 1,2-trans-DCVG > 2,2-cis-DCVG > 1,2-cis-DCVG, is likely to contribute to extrahepatic GSH-conjugation of TCE. Analysis of the products formed by a beta-lyase mimetic model showed that both 1,2-trans-DCVC and 1,2-cis-DCVC are converted to reactive products that form cross-links between the model nucleophile 4-(4-nitrobenzyl)-pyridine (NBP) and thiol-species. No NBP-alkylation was observed with 2,2-DCVC corresponding to its low cytotoxicity and mutagenicity. The lower activity of GSH-conjugation of TCE by human liver fractions, in combination with the lower fraction of potential nephrotoxic and mutagenic 1,2-DCVG-isomers, suggest that humans are at much lower risk for TCE-associated nephrotoxic effects than rats

Exposure to cis- and trans-regioisomers of S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)-glutathione result in quantitatively and qualitatively different cellular effects in RPTEC/TERT1 cells

Bioactivation of trichloroethylene (TCE) via glutathione conjugation is associated with several adverse effects in the kidney and other extrahepatic tissues. Of the three regioisomeric conjugates formed, S-(1,2-trans-dichlorovinyl)-glutathione (1,2-trans-DCVG), S-(1,2-cis-dichlorovinyl)-glutathione and S-(2,2-dichlorovinyl)-glutathione, only 1,2-trans-DCVG and its corresponding cysteine-conjugate, 1,2-trans-DCVC, have been subject to extensive mechanistic studies. In the present study, the metabolism and cellular effects of 1,2-cis-DCVG, the major regioisomer formed by rat liver fractions, and 1,2-cis-DCVC were investigated for the first time using RPTEC/TERT1-cells as in vitro renal model. In contrast to 1,2-trans-DCVG/C, the cis-regioisomers showed minimal effects on cell viability and mitochondrial respiration. Transcriptomics analysis showed that both 1,2-cis-DCVC and 1,2-trans-DCVC caused Nrf2-mediated antioxidant responses, with 3 µM as lowest effective concentration. An ATF4-mediated integrated stress response and p53-mediated responses were observed starting from 30 µM for 1,2-trans-DCVC and 125 µM for 1,2-cis-DCVC. Comparison of the metabolism of the DCVG regioisomers by LC/MS showed comparable rates of processing to their corresponding DCVC. No detectable N-acetylation was observed in RPTEC/TERT1 cells. Instead, N-glutamylation of DCVC to form N-γ-glutamyl-S-(dichlorovinyl)-L-cysteine was identified as a novel route of metabolism. The results suggest that 1,2-cis-DCVC may be of less toxicological concern for humans than 1,2-trans-DCVC, considering its lower intrinsic toxicity and lower rate of formation by human liver fractions.</p

Extending the Impact of RAC1b Overexpression to Follicular Thyroid Carcinomas

RAC1b is a hyperactive variant of the small GTPase RAC1 known to be a relevant molecular player in different cancers. Previous studies from our group lead to the evidence that its overexpression in papillary thyroid carcinoma (PTC) is associated with an unfavorable prognosis. In the present study, we intended to extend the analysis of RAC1b expression to thyroid follicular neoplasms and to seek for clinical correlations. RAC1b expression levels were determined by RT-qPCR in thyroid follicular tumor samples comprising 23 follicular thyroid carcinomas (FTCs) and 33 follicular thyroid adenomas (FTAs). RAC1b was found to be overexpressed in 33% of carcinomas while no RAC1b overexpression was documented among follicular adenomas. Patients with a diagnosis of FTC were divided into two groups based on longitudinal evolution and final outcome. RAC1b overexpression was significantly associated with both the presence of distant metastases (P = 0.01) and poorer clinical outcome (P = 0.01) suggesting that, similarly to that previously found in PTCs, RAC1b overexpression in FTCs is also associated with worse outcomes. Furthermore, the absence of RAC1b overexpression in follicular adenomas hints its potential as a molecular marker likely to contribute, in conjunction with other putative markers, to the preoperative differential diagnosis of thyroid follicular lesions

Looking out for cancer stem cells' properties: the value-driving role of CD44 for personalized medicines

The expression of CD44 tags cells with stemness-associated properties (cancer initiating cells or cancer stem cells - CSC). This membrane glycoprotein with a cytoplasmic domain indirectly associated with the cellular cytoskeleton, has a crucial role in tumorigenesis. The CD44 receptor enables the cell to respond to changes in tumor microenvironment, promoting several signaling events related to tumor initiation, progression and fixation in distant host tissues. Although the contribution of this transmembrane protein in gene regulation remains unclear, its overexpression in adenocarcinomas, mostly supported by microRNA (miR)-mediated upregulation of target mRNA, is widely accepted. Herein, we gather the evidence that CD44 is one of the most predominant markers of malignant cells and may be found in diverse phenotypes associated with tumor progression. Additionally, CD44 tumor receptors were found to have different roles at a transcriptional level. Thus, innovative therapeutic strategies should rely heavily on its metastasis-promoting ability. Furthermore, the concept of selectively targeting cell sub-populations may be used to develop specific therapeutic and/or diagnostic systems. An approach based on targeting CD44⁺ cells might provide a strategy to design guided-therapeutic systems against multiple malignant cells including putative CSC