Fucoxanthin alleviates the cytotoxic effects of cadmium and lead on a human osteoblast cell line

Abstract

Objective: Cadmium (Cd) and lead (Pb) are non-biodegradable heavy metals (HMs) that persistently contaminate ecosystems and accumulate in bones, where they exert harmful effects. This study aimed to investigate the protective effect of fucoxanthin (FX) against the chemical toxicity induced by Cd and Pb in human bone osteoblasts in vitro, using various biochemical and molecular assays.

Methods: The effect of metals and FX on osteoblasts viability was assayed by MTT, then the effect of Pb, Cd, and FX on the cells' mitochondrial parameters was studied via assays for ATP, mitochondrial membrane potential (MMP), mitochondrial complexes, and lactate production. Also, the effect of metals on oxidative stress was assessed by reactive oxygen species, lipid peroxidation and antioxidant enzymes assays. Also the effect of FX and metals on apoptosis caspases and related genes was assessed.

Results: When Cd and Pb were added to human osteoblast cultures at concentrations ranging from 1-20 μM for 72 h, they significantly reduced osteoblast viability in a time and concentration-dependent manner. The cytotoxic effect of Cd on osteoblasts was greater than that of Pb, with estimated EC50 of 8 and 12 μM, respectively, after 72 h of exposure. FX (10 and 20 μM) alleviated the cytotoxicity of the metals. Bioenergetics assays, including ATP, MMP, and mitochondrial complexes I and III activities, revealed that HMs at 1 and 10 μM concentrations inhibited cellular bioenergetics after 72 h of exposure. Cd and Pb also increased lipid peroxidation and reactive oxygen species while reducing catalase and superoxide dismutase antioxidant activities and oxidative stress-related genes. This was accompanied by increased caspases -3, -8, and - 9 and Bax/bCl-2 ratio. Co-treatment with FX (10 and 20 μM) mitigated the disruption of bioenergetics, oxidative damage, and apoptosis induced by the metals, showing a concentration-dependent pattern to varying extents.

Conclusion: These findings strongly support the role of FX in managing toxicities induced by environmental pollutants in bones and in addressing bone diseases associated with molecular bases of oxidative stress, apoptosis, and bioenergetic disruption.

Keywords: antioxidants; bone; cadmium; fucoxanthin; lead; osteoblasts; redox stress.

Fig. 1

The cytotoxic effect of different concentrations of lead (Pb) and cadmium (Cd) (1, 5, 10, and 20 μM) and fucoxanthin (FX) (5, 10, 20, and 40 μM) on cultured human osteoblasts 72 h after exposure (Fig. 1A). Figures 1B and D Demonstrate the protective effect of FX (10 and 20 μM) in reducing the cytotoxic effects of Pb and Cd at concentrations of 1 and 10 μM on human osteoblasts 72 h post-exposure. FX significantly reduced the cytotoxic effects of both metals to varying degrees. ^*P-value <0.05, ^**P-value <0.01, and ^***P-value <0.001.

Fig. 2

The effect of lead (Pb) and cadmium (Cd) at concentrations 1 and 10 μM and fucoxanthin (FX) (10 and 20 μM), 72 h post-exposure, on procollagen type I peptide cellular level (Fig. 2A and B) and culture media level (Fig. 2C and D), osteocalcin production (Fig. 2E and F), and alkaline phosphatase (ALP) (Fig. 2G and H) production of the cultured human osteoblasts. Data showed that both metals significantly inhibited the secretory functions of the treated cells in the three assays in a concentration-dependent pattern. FX significantly counteracts the metal-induced secretory dysfunction of the treated cells. Significance was evaluated by one-way ANOVA with Dunnett’s post-test comparing the outcomes in the presence and absence of FX. ^*P-value <0.05, ^**P-value <0.01, and ^***P-value <0.001.

Fig. 3

The effect of lead (Pb) and cadmium (Cd) at concentrations 1 and 10 μM and fucoxanthin (FX) (10 and 20 μM), 72 h post-exposure, on the cellular bioenergetics of the cultured human osteoblasts. The cells were treated with the metals alone or co-treated with the metals and FX for 72 h. ATP (Fig. 3A and B), mitochondrial membrane potential (MMP) (Fig. 3C and D), mitochondrial complex I (MCI) (Fig. 3F and D), mitochondrial complex III (MCIII) (fig. 3G and H), and lactate production assays (Fig. 3I and J) revealed that both metals significantly inhibited the treated cells’ bioenergetics in a concentration-dependent pattern. Cd showed more inhibitory effects in all assays. FX significantly improved the metals-treated cells’ ATP production, MMP, MCI, and MCIII activities with a significant decrease in lactate production. Significance was evaluated by one-way ANOVA with Dunnett’s post-test comparing the outcomes in the presence and absence of FX. ^*P-value <0.05, ^**P-value <0.01, and ^***P-value <0.001.

Fig. 4

The effect of lead (Pb) and cadmium (Cd) at concentrations 1 and 10 μM and fucoxanthin (FX) (10 and 20 μM), 72 h post-exposure, on the redox status of the cultured human osteoblasts. The cells were treated with the metals alone or co-treated with the metals and FX for 72 h. reactive oxygen species (ROS) (Fig. 4A and B), lipid peroxidation thiobarbituric acid (TBARS) product (Fig. 4C and D), antioxidant enzyme catalase (CAT) activity (Fig. 4E and F), superoxide dismutase (SOD) activity (Fig. 4G, 3H) were applied. Data showed that both metals significantly incased ROS and TBARS production with a significant decrease in CAT and SOD antioxidant activities in a concentration-dependent pattern. FX significantly counteracts the metals-induced oxidative damage and alleviates the inhibitory effect of the metals on CAT and SOD to variable extents. Significance was evaluated by one-way ANOVA with Dunnett’s post-test comparing the outcomes in the presence and absence of FX. ^*P-value <0.05, ^**P-value <0.01, and ^***P-value <0.001.

Fig. 5

The effect of lead (Pb) and cadmium (Cd) at concentrations 1 and 10 μM and fucoxanthin (FX) (10 and 20 μM), 72 h post-exposure, on the redox related genes expression of the cultured human osteoblasts. The cells were treated with the metals alone or co-treated with the metals and FX for 72 h. the Nrf2 (Fig. 5A and B), HO-1 (Fig. 5C and D), antioxidant enzyme catalase (CAT) (Fig. 5E and F), superoxide dismutase (SOD) (Fig. 5G and H) assays were applied. Data showed that both metals significantly decreased the expression of Nrf2, HO-1, CAT, and SOD genes in a concentration-dependent pattern. FX significantly counteracts the metals-induced inhibition of the expression of the antioxidant genes to variable extents. Significance was evaluated by one-way ANOVA with Dunnett’s post-test comparing the outcomes in the presence and absence of FX. ^*P-value <0.05, ^**P-value <0.01, and ^***P-value <0.001.

Fig. 6

The effect of lead (Pb) and cadmium (Cd) at concentrations 1 and 10 μM, and fucoxanthin (FX) (10 and 20 μM), 72 h post-exposure, on caspases −3 (Fig. 6A and6B), −8 (Fig. 6C and D), and − 9 (Fig. 6E and F) activities, Bax/Bcl2 genes expression ratio (Fig. 6G and H), in the cultured human osteoblasts. Data showed that both metals significantly inhibited the secretory functions of the treated cells in the three assays in a concentration-dependent pattern. FX significantly counteracts the metal-induced secretory dysfunction of the treated cells. Significance was evaluated by one-way ANOVA with Dunnett’s post-test comparing the outcomes in the presence and absence of FX. ^*P-value <0.05, ^**P-value <0.01, and ^***P-value <0.001.