H9c2(2-1)-based sulforhodamine B assay as a possible alternative in vitro platform to investigate effluent and metals toxicity on fish

Rodrigues, Elsa T.; Pardal, Miguel Ângelo; Pereira, Eduarda; Monteiro, Joana F.; Certal, Ana C.; Oliveira, Paulo J.

doi:10.1016/j.chemosphere.2021.130009

Cited by 5 publications

(6 citation statements)

References 28 publications

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“…To prepare the exposure solutions, 4.0 mL of cell culture medium was added to each lyophilised sample, and a sonicator (Branson Ultrasonics, 3510E-DTH) was used for 2 min to promote homogenisation. Then, in line with our two related studies (Rodrigues et al, 2021 ; Rodrigues et al, 2020a ), in the highest concentration well, 400 μL of the well medium was removed and replaced with 900 μL of exposure solution, and then serial dilutions were applied, making a test concentration range of 35.2–4,500%. Three replicates prepared from independent cell cultures (true replicates) were maintained for each concentration, and four negative (cells with culture medium alone) and four positive (cells with 2% DMSO prepared in culture medium) controls were considered by replicate.…”

Section: Methodsmentioning

confidence: 73%

“…We have previously shown that the rat cardiomyoblast H9c2(2-1) cell-based sulforhodamine B (SRB) colorimetric assay is suitable to quantitatively estimate the biological component of effluent toxicity (Rodrigues et al, 2020a ) and as a promising in vitro method to replace fish lethal testing of municipal effluents, both in relative and absolute terms (Rodrigues et al, 2021 ). Since wastewater composition is highly variable as a function of time, day of the week and season, in a stepwise approach, the present study aims to confirm the potentiality of the H9c2(2-1) cell-based SRB assay to evaluate the temporal variability of municipal wastewater collected over a year.…”

Section: Intorudctionmentioning

confidence: 99%

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The H9c2(2-1) cell-based sulforhodamine B assay is a non-animal alternative to evaluate municipal wastewater quality over time

Rodrigues,

Pereira,

Oliveira

et al. 2023

Environ Monit Assess

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The present study validates the potential of the in vitro H9c2(2-1) cell-based sulforhodamine B (SRB) assay to evaluate the temporal variability of wastewater quality. The impact of effluent disposal on water quality and the efficiency of the wastewater treatment process were also assessed. To correlate standard analytical method results with in vitro results, a total of 16 physicochemical parameters, such as nutrients, pH, chemical oxygen demand, total suspended solids and metals, were determined in both raw and treated wastewater samples. Results revealed that the H9c2(2-1) cell-based SRB assay has an enormous potential to evaluate municipal wastewater quality over time and to discriminate influent and effluent toxic characteristics, as well as for water quality monitoring and surveillance of the efficacy of treatment processes. Finally, the gathered results alerted to the impact of phosphates in a biological system, leading us to recommend the selection of this parameter as a potential environmental health indicator.

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Section: Methodsmentioning

confidence: 73%

Section: Intorudctionmentioning

confidence: 99%

The H9c2(2-1) cell-based sulforhodamine B assay is a non-animal alternative to evaluate municipal wastewater quality over time

Rodrigues,

Pereira,

Oliveira

et al. 2023

Environ Monit Assess

Self Cite

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“…Cytotoxicity was assessed using SRB assay with modification [ 44 ]. In brief, 5 × 10 4 cells were seeded in a 24-well plate and cultured with complete medium containing 10% FBS for 24 h. After treatments, cells were fixed with 10% trichloroacetic acid, incubated with SRB for 30 min, and washed with 1% acetic acid.…”

Section: Methodsmentioning

confidence: 99%

Adenine Combined with Cisplatin Promotes Anticancer Activity against Hepatocellular Cancer Cells through AMPK-Mediated p53/p21 and p38 MAPK Cascades

et al. 2022

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Cisplatin has been widely used in cancer treatments. Recent evidence indicates that adenine has potential anticancer activities against various types of cancers. However, the effects of the combination of adenine and cisplatin on hepatocellular carcinoma (HCC) cells remain sketchy. Here, our objective was to elucidate the anticancer activity of adenine in combination with cisplatin in HCC cells and its mechanistic pathways. Cell viability and cell cycle progression were assessed by the SRB assay and flow cytometry, respectively. Apoptosis was demonstrated by PI/annexin V staining and flow cytometric analysis. Protein expression, signaling cascade, and mRNA expression were analyzed by Western blotting and quantitative RT-PCR, respectively. Our results showed that adenine jointly potentiated the inhibitory effects of cisplatin on the cell viability of SK-Hep1 and Huh7 cells. Further investigation showed that adenine combined with cisplatin induced higher S phase arrest and apoptosis in HCC cells. Mechanically, adenine induced AMPK activation, reduced mTOR phosphorylation, and increased p53 and p21 levels. The combination of adenine and cisplatin synergistically reduced Bcl-2 and increased PUMA, cleaved caspase-3, and PARP in HCC cells. Adenine also upregulated the mRNA expression of p53, p21, PUMA, and PARP, while knockdown of AMPK reduced the increased expression of these genes. Furthermore, adenine also induced the activation of p38 MAPK through AMPK signaling, and the inhibition of p38 MAPK reduced the apoptosis of HCC cells with exposure to adenine combined with cisplatin. Collectively, these findings reveal that the combination of adenine and cisplatin synergistically enhances apoptosis of HCC cells, which may be attributed to the AMPK-mediated p53/p21 and p38 MAPK cascades. It suggests that adenine may be a potential adjuvant for the treatment of HCC in combination with cisplatin.

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“…Kramberger et al [14] reviewed the data supporting the internal use of H. Italicum in humans, indicating that the plant extract showed low cytotoxicity to cell lines, including the Hep G2, N9, and S17 cells, after 72 h of exposure at tested concentrations up to 100 µg mL −1 . Despite the reported differences in cell line sensitivity, it is a fact that cell-based toxicity tests are an emerging alternative to conventional tests with aquatic organisms, which are costly and time-demanding and raise ethical issues; however, it is necessary to evaluate cell lines that show sensitivities comparable to those of aquatic species [85,86] or even similar sensitivity rankings to preliminarily categorize the toxicity of diverse chemicals. Therefore, more research including tests performed under similar experimental conditions is required to assess the suitability of cell-based assays as an alternative to conventional tests with aquatic organisms.…”

Section: Essential Oils Ecotoxicity Towards Aquatic Organismsmentioning

confidence: 99%

Lethal Toxicity of Thymus mastichina and Helichrysum italicum Essential Oils to Non-Target Aquatic Organisms: Tools to Screen Environmental Effects?

Afonso,

Nogueira,

Cavaleiro

et al. 2023

Water

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Essential oils (EOs) from Thymus mastichina (EO-thyme) and Helichrysum italicum (EO-curry) have wide commercial applications, but little is known about their ecotoxicity to aquatic life. We evaluated the lethal toxicity of both EOs toward standard freshwater (Daphnia. magna and Thamnocephalus platyurus) and saltwater (Artemia sp.) species. Dimethylsulfoxide was used as a solvent after establishing a maximum safe but effective concentration of 1% (v/v). EO-curry was significantly more toxic than EO-thyme (24–48 h LC50 values of 15.93–55.80 and of 84.78–153.0 mg L−1, respectively) for all species; sensitivity ratios ranged from threefold for D. magna (48 h) and Artemia sp. (24 h) to fivefold for T. platyurus (24 h). Artemia sp. was the least sensitive, and T. platyurus was the most sensitive species, although significantly more so than D. magna only to EO-curry. The second major compound in EO-thyme, β-pinene (5%), is more toxic to aquatic life than major compound 1,8-cineole (62%), although 1,8-cineole facilitates penetration of other EO constituents into crustaceans’ epidermis. Among the main compounds of EO-curry, only α-pinene (13%) is known to be toxic to aquatic organisms. However, minor compounds present in both EOs, like p-cymene (0.3–1.1%), also cause synergistic effects by enhancing the penetration of other EO constituents. Before any of these standard tests can be recommended for the ecotoxicity characterization and environmental management of EOs, their sensitivity to a wider range of EOs, at least from closely related families, needs to be assessed.

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H9c2(2-1)-based sulforhodamine B assay as a possible alternative in vitro platform to investigate effluent and metals toxicity on fish

Cited by 5 publications

References 28 publications

The H9c2(2-1) cell-based sulforhodamine B assay is a non-animal alternative to evaluate municipal wastewater quality over time

The H9c2(2-1) cell-based sulforhodamine B assay is a non-animal alternative to evaluate municipal wastewater quality over time

Adenine Combined with Cisplatin Promotes Anticancer Activity against Hepatocellular Cancer Cells through AMPK-Mediated p53/p21 and p38 MAPK Cascades

Lethal Toxicity of Thymus mastichina and Helichrysum italicum Essential Oils to Non-Target Aquatic Organisms: Tools to Screen Environmental Effects?

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