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Profiling transcriptomes of human SH-SY5Y neuroblastoma cells exposed to maleic acid

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ABSTRACT

Background: Maleic acid is a multi-functional chemical widely used in the field of industrial chemistry for producing food additives and food contact materials. As maleic acid may contaminate food by the release from food packages or intentional addition, it raises the concern about the effects of excessive dietary exposure to maleic acid on human health. However, the influence of maleic acid on human health has not been thoroughly studied. In silico toxicogenomics approaches have found the association between maleic acid and nervous system disease in human. The aim of this study is to experimentally explore the effects of maleic acid on human neuronal cells.

Methods: A microarray-based transcriptome profiling was performed to offer a better understanding of the effects of maleic acid on human health. Gene expression profiles of human neuroblastoma SH-SY5Y cells exposed to three concentrations of maleic acid (10, 50, and 100 μM) for 24 h were analyzed. Genes which were differentially expressed in dose-dependent manners were identified and further analyzed with an enrichment analysis. The expression profile of selected genes related to the inferred functional changes was validated using quantitative polymerase chain reaction (qPCR). Specific fluorescence probes were applied to observe the inferred functional changes in maleic acid-treated neuronal cells.

Results: A total of 316 differentially expressed genes (141 upregulated and 175 downregulated) were identified in response to the treatment of maleic acid. The enrichment analysis showed that DNA binding and metal ion binding were the significant molecular functions (MFs) of the neuronal cells affected by maleic acid. Maleic acid exposure decreased the expression of genes associated with calcium and thiol levels of the cells in a dose-dependent manner. The levels of intracellular calcium and thiol levels were also affected by maleic acid dose-dependent.

Discussion: The exposure to maleic acid is found to decrease the cellular calcium and thiol levels in human neuronal cells at both transcriptional and functional levels. This study reported the first transcriptomic profiling of human neuronal cells treated with maleic acid. It is also the first experimental validation of chemical effects predicted by in silico toxicogenomics approaches. The proposed approach may be useful in understanding the potential effects of other poorly characterized chemicals on human health.

No MeSH data available.


Attenuation of cellular calcium and thiol level by maleic acid in SH-SY5Y cells.SH-SY5Y cells were seeded in a 96-well plate overnight and then exposed to maleic acid (0.1–100 μM). (A) Cell viability was evaluated by MTS and the absorbance was read at wavelength 490 nm by a microplate reader. Intracellular calcium and thiols of SH-SY5Y cells were measured by Fluo-4 AM (B) and CMF-DA (C), respectively. The change in fluorescence was analyzed by a microplate reader. The data were expressed as the mean ± SE of quadruplicate cultures. Results were representative of three independent experiments. *p < 0.05 was significant compared to the control group (cell alone without maleic acid).
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fig-3: Attenuation of cellular calcium and thiol level by maleic acid in SH-SY5Y cells.SH-SY5Y cells were seeded in a 96-well plate overnight and then exposed to maleic acid (0.1–100 μM). (A) Cell viability was evaluated by MTS and the absorbance was read at wavelength 490 nm by a microplate reader. Intracellular calcium and thiols of SH-SY5Y cells were measured by Fluo-4 AM (B) and CMF-DA (C), respectively. The change in fluorescence was analyzed by a microplate reader. The data were expressed as the mean ± SE of quadruplicate cultures. Results were representative of three independent experiments. *p < 0.05 was significant compared to the control group (cell alone without maleic acid).

Mentions: According to our previous transcriptome and enrichment analysis, calcium binding was inferred to be affected by maleic acid. Among the differentially expressed genes, eight genes are associated with the GO term of calcium binding (GO:0005509) including S100A3, GNPTAB, CDH23, PCDHAC1, PADI1, CRACR2B, MAN1A2, and CDH8. In addition, GNRHR and GRIA1 among the differentially expressed genes are involved in the regulation of cellular calcium levels. Altogether, cellular calcium is potentially affected by maleic acid. To validate this finding, the kinetic change of cellular calcium and thiol levels by maleic acid was analyzed by specific fluorescence Fluo-4 and CMF-DA probes, respectively. To avoid measuring the direct cytotoxic effects of maleic acid on SH-SY5Y cells, we first evaluated the cell viability of maleic acid-treated SH-SY5Y cells by an MTS assay. Figure 3A showed that there was no significant change in cell viability after 48 h of maleic acid treatment. By contrast, maleic acid (0.1–100 μM) significantly decreased the cellular calcium level by 12–41%, determined by the change of fluorescence intensity of Fluo-4, in a concentration- and time-dependent manner (Fig. 3B). Maleic acid (1–100 μM) decreased the cellular thiol levels by 16–31% after 24 h treatment (Fig. 3C). The exact p-values of Fig. 3 were shown in Supplemental Information 2. These results indicated that maleic acid resulted in a loss of cellular calcium level earlier than glutathione depletion. These observations could give the evidence that maleic acid at the concentrations higher than 0.1 μM interferes cellular calcium homeostasis in human neuronal cells. The homeostasis of intracellular Ca2+ plays important roles in the regulation of physiological responses or controlling cell death (Demuro et al., 2005; Bading, 2013; Brini et al., 2014). Depletion of intracellular Ca2+ has been reported to induce SH-SY5Y cell death by activating caspase activation (McGinnis, Wang & Gnegy, 1999). Our data showed that maleic acid significantly attenuated intracellular Ca2+ levels without induction of cell death, indicating that other pathways might be involved and need to be further studied.


Profiling transcriptomes of human SH-SY5Y neuroblastoma cells exposed to maleic acid
Attenuation of cellular calcium and thiol level by maleic acid in SH-SY5Y cells.SH-SY5Y cells were seeded in a 96-well plate overnight and then exposed to maleic acid (0.1–100 μM). (A) Cell viability was evaluated by MTS and the absorbance was read at wavelength 490 nm by a microplate reader. Intracellular calcium and thiols of SH-SY5Y cells were measured by Fluo-4 AM (B) and CMF-DA (C), respectively. The change in fluorescence was analyzed by a microplate reader. The data were expressed as the mean ± SE of quadruplicate cultures. Results were representative of three independent experiments. *p < 0.05 was significant compared to the control group (cell alone without maleic acid).
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Related In: Results  -  Collection

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fig-3: Attenuation of cellular calcium and thiol level by maleic acid in SH-SY5Y cells.SH-SY5Y cells were seeded in a 96-well plate overnight and then exposed to maleic acid (0.1–100 μM). (A) Cell viability was evaluated by MTS and the absorbance was read at wavelength 490 nm by a microplate reader. Intracellular calcium and thiols of SH-SY5Y cells were measured by Fluo-4 AM (B) and CMF-DA (C), respectively. The change in fluorescence was analyzed by a microplate reader. The data were expressed as the mean ± SE of quadruplicate cultures. Results were representative of three independent experiments. *p < 0.05 was significant compared to the control group (cell alone without maleic acid).
Mentions: According to our previous transcriptome and enrichment analysis, calcium binding was inferred to be affected by maleic acid. Among the differentially expressed genes, eight genes are associated with the GO term of calcium binding (GO:0005509) including S100A3, GNPTAB, CDH23, PCDHAC1, PADI1, CRACR2B, MAN1A2, and CDH8. In addition, GNRHR and GRIA1 among the differentially expressed genes are involved in the regulation of cellular calcium levels. Altogether, cellular calcium is potentially affected by maleic acid. To validate this finding, the kinetic change of cellular calcium and thiol levels by maleic acid was analyzed by specific fluorescence Fluo-4 and CMF-DA probes, respectively. To avoid measuring the direct cytotoxic effects of maleic acid on SH-SY5Y cells, we first evaluated the cell viability of maleic acid-treated SH-SY5Y cells by an MTS assay. Figure 3A showed that there was no significant change in cell viability after 48 h of maleic acid treatment. By contrast, maleic acid (0.1–100 μM) significantly decreased the cellular calcium level by 12–41%, determined by the change of fluorescence intensity of Fluo-4, in a concentration- and time-dependent manner (Fig. 3B). Maleic acid (1–100 μM) decreased the cellular thiol levels by 16–31% after 24 h treatment (Fig. 3C). The exact p-values of Fig. 3 were shown in Supplemental Information 2. These results indicated that maleic acid resulted in a loss of cellular calcium level earlier than glutathione depletion. These observations could give the evidence that maleic acid at the concentrations higher than 0.1 μM interferes cellular calcium homeostasis in human neuronal cells. The homeostasis of intracellular Ca2+ plays important roles in the regulation of physiological responses or controlling cell death (Demuro et al., 2005; Bading, 2013; Brini et al., 2014). Depletion of intracellular Ca2+ has been reported to induce SH-SY5Y cell death by activating caspase activation (McGinnis, Wang & Gnegy, 1999). Our data showed that maleic acid significantly attenuated intracellular Ca2+ levels without induction of cell death, indicating that other pathways might be involved and need to be further studied.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Maleic acid is a multi-functional chemical widely used in the field of industrial chemistry for producing food additives and food contact materials. As maleic acid may contaminate food by the release from food packages or intentional addition, it raises the concern about the effects of excessive dietary exposure to maleic acid on human health. However, the influence of maleic acid on human health has not been thoroughly studied. In silico toxicogenomics approaches have found the association between maleic acid and nervous system disease in human. The aim of this study is to experimentally explore the effects of maleic acid on human neuronal cells.

Methods: A microarray-based transcriptome profiling was performed to offer a better understanding of the effects of maleic acid on human health. Gene expression profiles of human neuroblastoma SH-SY5Y cells exposed to three concentrations of maleic acid (10, 50, and 100 &mu;M) for 24 h were analyzed. Genes which were differentially expressed in dose-dependent manners were identified and further analyzed with an enrichment analysis. The expression profile of selected genes related to the inferred functional changes was validated using quantitative polymerase chain reaction (qPCR). Specific fluorescence probes were applied to observe the inferred functional changes in maleic acid-treated neuronal cells.

Results: A total of 316 differentially expressed genes (141 upregulated and 175 downregulated) were identified in response to the treatment of maleic acid. The enrichment analysis showed that DNA binding and metal ion binding were the significant molecular functions (MFs) of the neuronal cells affected by maleic acid. Maleic acid exposure decreased the expression of genes associated with calcium and thiol levels of the cells in a dose-dependent manner. The levels of intracellular calcium and thiol levels were also affected by maleic acid dose-dependent.

Discussion: The exposure to maleic acid is found to decrease the cellular calcium and thiol levels in human neuronal cells at both transcriptional and functional levels. This study reported the first transcriptomic profiling of human neuronal cells treated with maleic acid. It is also the first experimental validation of chemical effects predicted by in silico toxicogenomics approaches. The proposed approach may be useful in understanding the potential effects of other poorly characterized chemicals on human health.

No MeSH data available.