Temperature is a key environmental driver of hepatic physiology in ectotherms, and three-dimensional (3D) fish liver models may serve as an ethically advantageous platform to investigate warming effects under controlled conditions while reducing the need for experimental animals. Despite their increasing use in toxicology, their application to assess climate-relevant temperature effects on liver function remains limited. This study investigated the temporal effects of a warming scenario on primary hepatocyte spheroids from juvenile brown trout (Salmo trutta), a bioindicator species. The study explores how a + 3 °C increase can affect spheroid development and maintenance, as well as its impact on metabolic activity, cell proliferation and death, and morphology over time. Spheroids were maintained at 18 °C and 21 °C for 25 days and analysed at five time points using metabolic, morphometric, immunocytochemical, and ultrastructural approaches. Mitochondrial metabolic activity, assessed by resazurin reduction, showed no significant temperature-related differences. In contrast, warming accelerated spheroid formation and produced larger spheroids. Proliferative activity, assessed by proliferating cell nuclear antigen (PCNA) immunostaining, was significantly reduced at 21 °C, while caspase-3 levels remained unchanged, indicating no increase in apoptosis. The autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3A/B) showed lower immunoreactivity at 21 °C, with no temporal variation. Ultrastructural analysis revealed preserved hepatocyte integrity at both temperatures and abundant cytoplasmic dense bodies consistent with autolysosomal structures, which increased over time. Overall, a realistic warming scenario altered growth dynamics and cellular morphology. Further, this data reinforces that 3D fish liver models are viable alternative systems for assessing climate-driven effects.

Characterization of sex and biometry of fish donors for primary cell culture is often overlooked. Consequently, the influence of donor biological variability—such as reproductive status—on basal estrogenic expression in fish hepatocytes remains underexplored in toxicological studies. This study examines whether inherent differences among 1-year-old juvenile brown trout (Salmo trutta) are reflected in estrogenic signatures in liver tissue and the corresponding primary hepatocyte cultures from the same individuals. To our knowledge, no previous research has systematically assessed interindividual variation in estrogenic markers in brown trout primary hepatocytes. To address this, we evaluated the gene and protein expression of four classical estrogenic targets: vitellogenin, zona pellucida protein, estrogen receptor alpha, and estrogen receptor beta. No statistically significant differences in basal expression levels were observed across male and female donors in either hepatocytes or liver tissue. Nonetheless, interindividual variability was evident, reflecting natural biological differences in gonadal development and physiological status. The findings highlight the need to document and control donor sex, reproductive status, and individual variability when selecting fish for hepatocyte culture, even when sex differences in basal expression are not statistically significant, thereby providing a crucial baseline for toxicological experiments using primary trout hepatocytes.

Polycyclic aromatic hydrocarbons (PAHs) and global warming impact aquatic ecosystems, eventually interacting. Monolayer (2D) cultures of cell lines, such as the rainbow trout liver RTL-W1, are employed for unveiling toxicological effects in fish. Nonetheless, three-dimensional (3D) models constitute an alternate paradigm, better emulating in vivo responses. Here, ultra-low attachment (ULA) plates were used to generate ten-day-old RTL-W1 spheroids for exposure to a control, a solvent control (0.1% DMSO) and the model PAH benzo[k]fluoranthene (BkF) at 10 and 100 nM and at 18 and 23 °C (thermal stress). After a 4-day exposure, spheroids were analyzed for viability (alamarBlue and lactate dehydrogenase), biometry (area, diameter and sphericity), histocytology (optical and electron microscopy), and mRNA levels of the detoxification-related genes cytochrome P450 (CYP)1A, CYP3A27, aryl hydrocarbon receptor (AhR), glutathione S-transferase (GST), uridine diphosphate–glucuronosyltransferase (UGT), catalase (CAT), multidrug resistance-associated protein 2 (MRP2) and bile salt export protein (BSEP). Immunocytochemistry (ICC) was used to assess CYP1A protein expression. Neither temperature nor BkF exposure altered the spheroids’ viability or biometry. BkF modified the cell’s ultrastructure. The expression of CYP1A was augmented with both BkF concentrations, while AhR’s increased at the higher concentration. The CYP1A protein showed a dose-dependent increase. Temperature and BkF concurrently modelled UGT’s expression, which increased in the 100 nM condition at 23 °C. Conversely, CYP3A27, MRP2, and BSEP expressions lowered at 23 °C. CAT and GST mRNA levels were uninfluenced by either stressor. Overall, BkF and temperature impacted independently or interactively in RTL-W1 spheroids. These seem to be useful novel tools for studying the liver-related effects of temperature and PAHs.

Feline infectious peritonitis (FIP) is caused by a feline coronavirus (FCoV) and it is one of the most important viral diseases of cats. Recently, new drugs have been developed and used to face the inexorable fatal prognosis of FIP when untreated. More than ever, a definitive ante-mortem diagnosis is crucial to identify the cats that would benefit from antiviral treatment and to avoid euthanasia of unaffected animals. Identification of FCoV in macrophages using immunohistochemistry (IHC) is the gold standard method for diagnosing FIP. In this pilot study, the role of IHC applied to cell tube blocks (CTBs) of effusion fluids of cats clinically suspected to have FIP was assessed. A prospective cohort of seven cats was enrolled. IHC results in CTBs was positive in six out seven cases. Necropsy, histology and IHC in organs/tissues confirmed FIP infection in all the seven cases. The false negative in a CTB corresponded to a case of bacterial peritonitis secondary to FIP. As proof-of-concept, CTBs are valuable as a non-invasive technique for a definitive ante-mortem/clinical diagnosis of FIP and it is recommended to obtain CTBs from effusion fluids of all suspected FIP cases.

Immunolabeling on Romanowsky-stained cytology (RSC) slides can be used, although there is limited evidence of its suitability for phenotyping canine and feline lymphomas. A comparison with matched cell blocks (CB) is missing. Immunolabeling on RSC and CB was compared for lymphoid markers (CD3 and PAX5) in 53 lymphomas and 4 chylous effusions from dogs and cats. The influence of pre-analytical variables (species, time of archive, type of specimens and coverslipping) and the interobserver agreement among the 2 observers was assessed. Fewer CD3+ lymphocytes were identified in RSC, while the PAX5 positivity by RSC and CB had a substantial agreement. Immunodetection of CD3 and the diagnosis of a T-cell population on RSC was more difficult. Lower intensity and higher background were noted in RSC. Immunophenotyping was inconclusive in 54% RSC and 19% CB. The interobserver reproducibility of immunophenotyping on CB was substantial, being higher than in RSC. The immunolabeling performance on the RSC of effusion and feline samples was unsatisfactory. The detection of lymphoid markers, especially membranous antigens in retrospective RSC, is affected by the pre-analytical variables: species, time of the archive, and type of specimens. CB are a more consistent type of sample for immunophenotyping purposes.

Triple-negative breast cancer (TNBC) represents an aggressive subtype of breast cancer (BC) with a typically poorer prognosis than other subtypes of BC and limited therapeutic options. Therefore, new drugs would be particularly welcome to help treat TNBC. Preussin, isolated from the marine sponge-associated fungus, Aspergillus candidus, has shown the potential to reduce cell viability and proliferation as well as to induce cell death and cell cycle arrest in 2D cell culture models. However, studies that better mimic the tumors in vivo, such as 3D cell cultures, are needed. Here, we studied the effects of preussin in the MDA-MB-231 cell line, comparing 2D and 3D cell cultures, using ultrastructural analysis and the MTT, BrdU, annexin V-PI, comet (alkaline and FPG modified versions), and wound healing assays. Preussin was found to decrease cell viability, both in 2D and 3D cell cultures, in a dose-dependent manner, impair cell proliferation, and induce cell death, therefore excluding the hypothesis of genotoxic properties. The cellular impacts were reflected by ultrastructural alterations in both cell culture models. Preussin also significantly inhibited the migration of MDA-MB-231 cells. The new data expanded the knowledge on preussin actions while supporting other studies, highlighting its potential as a molecule or scaffold for the development of new anticancer drugs against TNBC.

Veterinary cytology faced a remarkable evolution in the last 15 years, in part due to increase recognition of the advantages of the cytology by veterinary clinicians. Simultaneously, there has been a growing awareness by the owners about the importance of a complete diagnostic workup aimed at defining a proper treatment protocol. With the extended use of cytology, challenging diagnostic cases are more frequent, and more clinically useful answers are requested. In this scenario, the use of cytology specimens to perform ancillary techniques is a valid approach. Rather than being simply archived, cytology slides can be a valuable source and a good platform to carry out cytochemistry, immunocytochemistry, and molecular techniques. Therefore, several diagnostic techniques can be applied in tiny samples, thus following the “doing more with less” principle. The aim of this approach is to refine the cytologic diagnosis and provide additional prognostic and therapeutic information. Herein, we detailed this principle in veterinary cytology and reviewed the use of cytology specimens for ancillary techniques as a single procedure, i.e., using the whole slide, or multiple procedures, i.e., multiple procedures applied in the same slide.

Three-dimensional cell cultures may better mimic avascular tumors. Yet, they still lack characterization and standardization. Therefore, this study aimed to (a) generate multicellular aggregates (MCAs) of four breast cell lines: MCF7, MDA-MB-231, and SKBR3 (tumoral) and MCF12A (non-tumoral) using ultra-low attachment (ULA) plates, (b) detail the methodology used for their formation and analysis, providing technical tips, and (c) characterize the MCAs using morphometry, qualitative cytology (at light and electron microscopy), and quantitative immunocytochemistry (ICC) analysis. Each cell line generated uniform MCAs with structural differences among cell lines: MCF7 and MDA-MB-231 MCAs showed an ellipsoid/discoid shape and compact structure, while MCF12A and SKBR3 MCAs were loose, more flattened, and presented bigger areas. MCF7 MCAs revealed glandular breast differentiation features. ICC showed a random distribution of the proliferating and apoptotic cells throughout the MCAs, not fitting in the traditional spheroid model. ICC for cytokeratin, vimentin, and E-cadherin showed different results according to the cell lines. Estrogen (ER) and progesterone (PR) receptors were positive only in MCF7 and human epidermal growth factor receptor 2 (HER-2) in SKBR3. The presented characterization of the MCAs in non-exposed conditions provided a good baseline to evaluate the cytotoxic effects of potential anticancer compounds.

Seaweed bioactive compounds have shown anticancer activities in in vitro and in vivo studies. However, tests remain limited, with conflicting results, and effects in combination with anticancer drugs are even scarcer. Here, the cytotoxic effects of five seaweed compounds (astaxanthin, fucoidan, fucosterol, laminarin, and phloroglucinol) were tested alone and in combination with anticancer drugs (cisplatin-Cis; and doxorubicin-Dox), in breast cell lines (three breast cancer (BC) subtypes and one non-tumoral). The combinations revealed situations where seaweed compounds presented potentiation or inhibition of the drugs' cytotoxicity, without a specific pattern, varying according to the cell line, concentration used for the combination, and drug. Fucosterol was the most promising compound, since: (i) it alone had the highest cytotoxicity at low concentrations against the BC lines without affecting the non-tumoral line; and (ii) in combination (at non-cytotoxic concentration), it potentiated Dox cytotoxicity in the triple-negative BC cell line. Using a comparative approach, monolayer versus 3D cultures, further investigation assessed effects on cell viability and proliferation, morphology, and immunocytochemistry targets. The cytotoxic and antiproliferative effects in monolayer were not observed in 3D, corroborating that cells in 3D culture are more resistant to treatments, and reinforcing the use of more complex models for drug screening and a multi-approach that should include histological and ICC analysis.

Fucoxanthin (Fx) is a carotenoid derived from marine organisms that exhibits anticancer activities. However, its role as a potential drug adjuvant in breast cancer (BC) treatment is still poorly explored. Firstly, this study investigated the cytotoxic effects of Fx alone and combined with doxorubicin (Dox) and cisplatin (Cis) on a panel of 2D-cultured BC cell lines (MCF7, SKBR3 and MDA-MB-231) and one non-tumoral cell line (MCF12A). Fucoxanthin induced cytotoxicity against all the cell lines and potentiated Dox cytotoxic effects towards the SKBR3 and MDA-MB-231 cells. The combination triggering the highest cytotoxicity (Fx 10 µM + Dox 1 µM in MDA-MB-231) additionally showed significant induction of cell death and genotoxic effects, relative to control. In sequence, the same combination was tested on 3D cultures using a multi-endpoint approach involving bioactivity assays and microscopy techniques. Similar to 2D cultures, the combination of Fx and Dox showed higher cytotoxic effects on 3D cultures compared to the isolated compounds. Furthermore, this combination increased the number of apoptotic cells, decreased cell proliferation, and caused structural and ultrastructural damages on the 3D models. Overall, our findings suggest Fx has potential to become an adjuvant for Dox chemotherapy regimens in BC treatment.

Brown trout is an environmental freshwater sentinel species and is economically important for recreational fishing and aquaculture. Despite that, there is limited knowledge regarding morpho-physiological variations in adults throughout the reproductive cycle. Thus, this study aimed to analyze the fitness and gonadal maturation of cultured adult brown trout in four reproductive phases (spawning capable—December, regressing—March, regenerating—July, and developing—November). The systematic evaluation of males and females was based on biometric, biochemical, and hormonal parameters, along with a histomorphological grading of gonads and the immunophenotype location of key steroidogenic enzymes. The total weight and lengths reached the lowest levels in December. Gonad weights were higher in December and November, while the opposite pattern was found for liver weights. The lowest levels of cholesterol and total protein were also noted during those stages. The 11-ketotestosterone (11-KT) and testosterone (T) for males, and estradiol (E2) and T for females, mostly explained the hormonal variations. The immunohistochemistry of cytochrome P450c17 (CYP17-I), aromatase (CYP19), and 17β-hydroxysteroid dehydrogenase (17β-HSD) showed sex and site-specific patterns in the distinct reproductive phases. The sex- and season-specific changes generated discriminative multi-parameter profiles, serving as a tool for environmental and aquaculture surveys.

Triple-negative breast cancer (TNBC) has the poorest BC prognosis, chemotherapy being the mainstream treatment. Prior studies revealed potential synergistic effects of natural compounds with drugs, such as doxorubicin (Dox), frequently used for TNBC. Fucosterol (Fct), a phytosterol of brown seaweeds, because of its antioxidant and antitumor effects, is one of these promising compounds. Using a TNBC cell line (MDA-MB-231), we aimed to test the effects of Fct alone and in combination with Dox on cell viability and proliferation, in monolayer and three-dimensional (3D) cultures. At this stage of the research, data demonstrated that Fct (5 µM) alone did not affect cell viability and proliferation. In monolayer, Dox (≥1 µM) decreased cell viability and proliferation, while in 3D only cell viability was affected at Dox 5 µM. The combination of Fct/Dox (5/0.1 µM), in monolayer, significantly decreased cell viability and proliferation, differing from the control and both compounds alone; hence, this suggests that Fct enhanced the Dox effect. These promising Fct effects in monolayer were not observed in 3D. We suggest that Fct may increase Dox effects, under certain conditions. Our results corroborate other studies reporting more treatment resistance of cells in 3D culture, reinforcing the need to use more complex models for more realistic drug screening.

Fish batches are often transported from aquacultures to laboratory facilities, and this fact triggers a pre-stressed condition that can cause alterations in both physiological and behavioural status of animals. The acclimation before fish trials is highly recommended to ideally ensure basal levels for the species, but there is still limited information concerning the period of time and parameters to be included for the evaluation. Facing those facts, brown trout (Salmo trutta f. fario) juveniles subjected to an acute transport (around 2 h) were here tested during 42 days post-stress (at 12 h, 7, 14, 28 and 42 days), in order to: 1) define a multi-parameter approach, based on biochemical, physical, morphological, nutritional and behavioural parameters that best characterize the fish acclimation and welfare status; and (2) propose an adequate acclimation period for brown trout juveniles after transportation to new housing conditions. Behavioural changes were noted with an overall stabilization at the 17th day. Fish tended to occupy preferentially the bottom of the tank, a smaller number of tank sections, decreased the swimming activity and the number of abnormal movements. Reaction time to food decreased until it was instantaneous, and the reaction to food gradually changed over time, so that on the 1st day there was no reaction and, from the 30th day, most of the fish moved continuously to reach the food. Feed intake did not occur on the 1st day, and then increased gradually. Despite that, there were no differences in fish condition and hepatosomatic indices over time. In general, glucose was relatively stable along the assay, and cholesterol decreased at the 7th and 14th days, compared to 12 h, possibly as a consequence of the high metabolic demand induced by stress. Cortisol varied from 21,841.9 pg/mL (12 h) to 2162.2 pg/mL (42 day). As for cortisol, the volume-weighted nuclear volume of interrenal cells was not significantly distinct between sampling days, but a minimum of 96.33 mu m(3) was reached at 28 days. Higher haemoglobin values were obtained at 28th and 42th days, in relation to 12 h and the 14th day. Triglycerides, potassium and haematocrit did not change. The quantitative skin analyses (fragments and histological sections) showed a reduction in the pigmented area, with stabilization at 14th day. Acclimation of brown trout juveniles should be monitored using a multi-parameter evaluation including behavioural, biochemical, nutritional and morphological analyses. Overall, it is recommended an acclimation period of at least 28 days.

Doxorubicin (Dox) is one of the most successful anticancer drugs in use. However, chemoresistance is one of the main limitations that patients face. Therefore, development of new strategies to improve the efficacy of Dox is needed. Marine-derived fungi are especially promising sources of new anticancer compounds. In this work, antitumor activity of crude ethyl extract of the cultures of the marine-derived fungus Neosartorya siamensis KUFA 0017 (NS), combined with Dox, was evaluated in six cancer cell lines. To evaluate possible mechanisms involved in the eventual improvement of Dox's cytotoxicity by NS extract, effects on DNA damage, cell death, ultrastructural modifications, and intracellular accumulation of Dox were assessed. The NS extract demonstrated a significant enhancement of Dox's cytotoxic activity in A549 cells, inducing DNA damage, cell death, and intracellular accumulation of Dox. Additionally, the cytotoxic effect of eight compounds, isolated from this extract, that is, 2,4-dihydroxy-3-methylacetophenone-(C1), nortryptoquivaline-(C2), chevalone C-(C3), tryptoquivaline H-(C4), fiscalin A-(C5), epi-fiscalin-C (C6), epi-neofiscalin A-(C7), and epi-fiscalin A-(C8), alone and combined with Dox was also evaluated in lung cancer cells. The cytotoxic effect of Dox was potentiated by all the isolated compounds (except C1) in A549 cells. Therefore, we concluded that NS extract potentiated cytotoxicity by inhibiting cell proliferation, increasing intracellular accumulation of Dox, and inducing cell death (possibly by an autophagic process). The isolated compounds also enhanced the activity of Dox, supporting the potential of this sort of combination. These data call for further studies to characterize drug interactions and underlying mechanisms.

Veterinary Clinical PathologyVolume 48, Issue 1 p. 9-10 LABORATORY MEDICINE "Now you see it, now you don't": The fate of mast cells in blood Paula Brilhante Simões, Paula Brilhante Simões INNO Veterinary Laboratory, University of Porto, Porto, PortugalSearch for more papers by this authorFernanda Malhão, Fernanda Malhão Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, PortugalSearch for more papers by this authorRicardo Marcos, Ricardo Marcos Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, PortugalSearch for more papers by this author Paula Brilhante Simões, Paula Brilhante Simões INNO Veterinary Laboratory, University of Porto, Porto, PortugalSearch for more papers by this authorFernanda Malhão, Fernanda Malhão Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, PortugalSearch for more papers by this authorRicardo Marcos, Ricardo Marcos Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, PortugalSearch for more papers by this author First published: 29 March 2019 https://doi.org/10.1111/vcp.12707Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume48, Issue1March 2019Pages 9-10 RelatedInformation

Breast cancer (BC) accounts for the most cancer-related deaths amongst women worldwide, implying an urgent need of finding new drugs more effective than those currently in use [1, 2]. Fucoxanthin (Fx) is a marine carotenoid derived from brown seaweed that has been showing antitumor effects on different cancer cell lines, mainly in 2D models [3]. However, 3D culture models have a better predictive capacity of in vivo cellular responses against cytotoxic compounds [4]. This study aimed to evaluate the potential anticancer effects of Fx versus Doxorubicin (Dox) (a conventional anticancer drug) in a panel of three BC cell lines, representative of different molecular subtypes (MCF-7, SKBR3 and MDA-MB-231), and in a non-tumoral BC cell line (MCF-12A), cultured under 2D and 3D conditions. Effects on cell viability, death, proliferation and DNA damage were targeted. Results from the 2D cultures showed that Dox and Fx caused cytotoxicity on all the cell lines. Cytotoxic activity of Dox involved mechanisms of cell death induction (in MCF-12A cells) and genotoxicity (in MDA-MB-231 cells), contrarily to Fx, where neither cell death nor genotoxic mechanisms were detected. Under 3D conditions, cells were less responsive to Dox and Fx, compared to 2D cultures. Antiproliferative effects were solely registered in SKBR3 cells exposed to Dox and Fx, and in MCF-12A cells exposed to Dox. The in vitro data revealed that the Fx may be a potential anticancer agent against BC cells, with differential effects according to the cell subtype. The data warrants further studies on the underlying anticancer mechanisms. Acknowledgments: We thank the fundamental financial support of the ICBAS of the UPorto, namely via its Laboratory of Histology and Embryology and Master Program in Environmental Contamination and Toxicology. This research was also partially supported by the Strategic Funding UID/Multi/04423/2019 through national funds provided by FCT and ERDF, in the framework of the program PT2020. [1] DeSantis, C.E., F. Bray, J. Ferlay, J. Lortet-Tieulent, B.O. Anderson, and A. Jemal, International Variation in Female Breast Cancer Incidence and Mortality Rates. Cancer Epidemiol Biomarkers Prev, 2015. 24(10): p. 1495-506. [2] Pádua, D., E. Rocha, D. Gargiulo, and A.A. Ramos, Bioactive compounds from brown seaweeds: Phloroglucinol, fucoxanthin and fucoidan as promising therapeutic agents against breast cancer. Phytochem Lett, 2015. 14: p. 91-98. [3] Kumar, S.R., M. Hosokawa, and K. Miyashita, Fucoxanthin: a marine carotenoid exerting anti-cancer effects by affecting multiple mechanisms. Mar Drugs, 2013. 11(12): p. 5130-47. [4] Santo, V.E., S.P. Rebelo, M.F. Estrada, P.M. Alves, E. Boghaert, and C. Brito, Drug screening in 3D in vitro tumor models: overcoming current pitfalls of efficacy read-outs. Biotechnol J, 2017. 12(1): p. 1600505.

Background Cell blocks and immunohistochemistry (IHC) are increasingly recognized as being complementary tools for cytologic diagnostics, especially for neoplastic diseases. Objectives The study aimed to evaluate the utility of cell tube block (CTB) IHC for refining the diagnosis of effusions in dogs and cats. Methods Cavitary effusions (n = 25) from dogs and cats classified by cytology as reactive, neoplastic, borderline (suspicious of neoplasia), and chylous were studied. CTB sections were stained with H&E, and immunostained with PAX‐5, CD3, pancytokeratin (CK), vimentin, and Wilms tumor 1 protein (WT1) antibodies, according to the cytologic diagnoses. A histologic case series of confirmed normal, reactive, and neoplastic mesothelium and several different carcinomas were included to test the utility of WT1 as a marker of mesothelial cells. Results CTBs had a layered appearance with reduced background staining. CD3 and PAX5 immunolabeling allowed immunophenotype assessment in all of the lymphoma cases. In carcinomatous effusions, neoplastic cells were CK‐positive, WT1‐negative, and vimentin‐negative (except for two cases). Wilms tumor 1 protein was positive in the nuclei of normal, reactive, and neoplastic mesothelial cells, and ovarian carcinomatous cells. Other carcinomas and lymphomas were negative. Conclusions CTBs are valuable tools to assist in making a diagnosis of cavitary effusions in dogs and cats, and WT1 is a promising marker to differentiate mesothelial from carcinomatous cells.

Preussin, a hydroxyl pyrrolidine derivative isolated from the marine sponge-associated fungus Aspergillus candidus KUFA 0062, displayed anticancer effects in some cancer cell lines, including MCF7. Preussin was investigated for its cytotoxic and antiproliferative effects in breast cancer cell lines (MCF7, SKBR3, and MDA-MB-231), representatives of major breast cancers subtypes, and in a non-tumor cell line (MCF12A). Preussin was first tested in 2D (monolayer), and then in 3D (multicellular aggregates), cultures, using a multi-endpoint approach for cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), resazurin and lactate dehydrogenase (LDH)) and proliferative (5-bromo-2′-deoxyuridine (BrdU)) assays, as well as the analysis of cell morphology by optical/electron microscopy and immunocytochemistry for caspase-3 and ki67. Preussin affected cell viability and proliferation in 2D and 3D cultures in all cell lines tested. The results in the 3D culture showed the same tendency as in the 2D culture, however, cells in the 3D culture were less responsive. The effects were observed at different concentrations of preussin, depending on the cell line and assay method. Morphological study of preussin-exposed cells revealed cell death, which was confirmed by caspase-3 immunostaining. In view of the data, we recommend a multi-endpoint approach, including histological evaluation, in future assays with the tested 3D models. Our data showed cytotoxic and antiproliferative activities of preussin in breast cancer cell lines in 2D and 3D cultures, warranting further studies for its anticancer potential.

The crosstalk between peroxisome proliferator-activated receptor α (PPARα) and estrogenic pathways are shared from fish to humans. Salmonid fish had an additional genome duplication, and two PPARα isoforms (PPARαBa and PPARαBb) were previously identified. Since a negative regulation between estrogen signaling and PPARα was described, a post-transcriptional gene silencing for PPARαBb was designed in primary brown trout hepatocytes. The aims of the study were to: (i) decipher the effects of PPARαBb knock-down on peroxisome morphology and on mRNA expression of potential target genes, and (ii) to assess the cross-interferences caused by an estrogenic compound (17α-ethinylestradiol – EE2) and a PPARα agonist (Wy-14,643 – Wy) using the established knock-down model. A knock-down efficiency of 70% was achieved for PPARαBb and its silencing significantly reduced the volume density of peroxisomes, but did not alter mRNA levels of the studied genes. Exposure to Wy did not change peroxisome morphology or mRNA expression, but under silencing conditions Wy rescued the volume density of peroxisomes to control levels, and increased acyl-coenzyme A oxidase 1-3l (Acox1-3l) mRNA. Exposure to EE2 caused a reduction of peroxisome volume density, but under silencing conditions this effect was abolished and ApoA1 mRNA level was diminished. The morphological alterations of peroxisomes by WY and EE2 demonstrated that obtained results are PPARαBb dependent, and suggest the regulation of unknown downstream targets of PPARαBb. In summary, PPARαBb is involved in the control of peroxisome size and/or number, which opens future opportunities to explore its regulation and molecular targets.