Relationship Between Intracellular Calcium And Reactive Oxygen Species On Sodium Fluoride-induced Injury In Human Neuroblastoma SH-SY5Y Cells

Endemic fluorosis which seriously impairs human health is prevant in the world. Excessive fluoride can cause widespread damage of predominately the skeletal systems and teeth as well as the structure and functions of the non-skeletal systems, such as brain, liver, kidney and spinal cord. Lots of studies have demonstrated that reactive oxygen species (ROS) may play an important role in the toxicological mechanisms induced by fluoride. Expermentals have shown that fluoride can cause an increase of intracellular calcium ([Ca²⁺]_i) level. Abnormal increase of [Ca²⁺]_i induces acute or chronic cell damage and death.Relationship between [Ca²⁺]_i and ROS is complicate and close. ROS can cause the injury of endoplasmic reticulum (ER) membrane, mitochondrion membrane and cell membrane which are reservoir pool of [Ca²⁺]_i. The injury of the membrane structure induces second distribution of [Ca²⁺]_i and inflow of extracellular calcium that cause the increase of [Ca²⁺]_i. [Ca²⁺]_i and ROS can induce mitochondrial permeability transition pore (MPTP) to open. On the one hand, MPTP's open causes ROS production. Furthermore MPTP's open induces rupture of mitochondrial membrane which will induce the calcium (Ca2+) releasing from intramembranous space. [Ca²⁺]_i regarding as second messenger and the increase of [Ca²⁺]_i can open many signal transduction path which may cause the production of ROS.The production of ROS and the increase of [Ca²⁺]_i play important roles in cell damage. So the relationship between them should get attention. However, at present the relationship between [Ca²⁺]_i and ROS in fluorosis is unclear. Which one is initiation? Which one is following? Are they causality or parallet? So far all those are rarely reported.Thus we treat human neuroblastoma SH-SY5Y cell as target and did some experiments in vitro to explore effects on levels of [Ca²⁺]_i, ROS and lactate dehydrogenae (LDH) induced by different dose sodium fluoride (NaF). Then we choose suitable NaF concentration to incubated with or without intracellular calcium-chelating compound (1,2-bis-(2-aminophenoxy)-ethane-N,N, N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester, BAPTA-AM), extracellular calcium chelator (ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, EGTA), and antioxidant (N-acetyl-L-cysteine, NAC) to observe the changes of [Ca²⁺]_i, ROS and LDH levels. The aims of the studies were to investigate the relationship between [Ca²⁺]_i and ROS and supply research data for further investigations about fluorosis mechanisms.This paper is consisted of two parts as followPart I Effects of fluoride on cytotoxicity, level of [Ca²⁺]_i and ROS in human neuroblastoma SH-SY5Y cells in vitroObjective To explore the effects of fluoride on cytotoxicity, [Ca²⁺]_i and ROS in SH-SY5Y cells in vitro and find the peak time of [Ca²⁺]_i and ROS. Methods SH-SY5Y cells were exposed to 20, 40 and 80μg/ml fluoride. [Ca²⁺]_i level was consecutively detected by Laser scanning confocal microscope (LSCM), LDH level in culture medium was measured by colorimetric method, and intracellular ROS level was detected by flow cytometrtl (FCM). In addition, 40μg/ml NaF was used to deteced changes of the levels of [Ca²⁺]_i and ROS at different exposed time. Results Comoared with control group, the levels of ROS and LDH in 40 and 80μg/ml NaF -treated groups were significatly higher (P<0.05, P<0.05, respectively). [Ca²⁺]_i levels of control group and 20μg/ml NaF-treated group were not obviously changed in 2 h. [Ca²⁺]_i level in 40μg/ml NaF -treated group began to rise slowly at about 5000 second, went to peak at about 6000 second and kept on to 10000 second, then began to descrease. [Ca²⁺]_i level in 80μg/ml NaF-treated group went to peak at about 2500 s and then decrease quickly. Compared with control group, [Ca²⁺]_i and ROS levels at different time were significatly higher (P<0.05) exclude ROS level at 3 h. The peak time of [Ca²⁺]_i and ROS levels were 3 h and 12 h, respectably. Conclusion NaF can induce the injury of cell membrane and the increase of [Ca²⁺]_i and ROS levels. The peak times of [Ca²⁺]_i and ROS levels were 3 h and 12 h, respectably.Part II Relationship between [Ca²⁺]_i and ROS on NaF-induced injury in human neuroblastoma SH-SY5Y cells in vitroObjective To investigate the relationship between [Ca²⁺]_i and ROS on NaF-induced injury in human neuroblastoma SH-SY5Y cells in vitro. Methods BAPTA-AM, EGTA, and NAC were combined with 40μg/ml NaF, the levels of [Ca²⁺]_i, ROS, LDH were measured at and 3 h, 12 h, and 24 h respectively. Results Compared with the 40μg/ml NaF-treated group, at 3 h BAPTA-AM decreased the [Ca²⁺]_i level, EGTA and NAC had no influences on [Ca²⁺]_i level respectably; At 12 h BAPTA-AM and NAC decreased the ROS level, EGTA had no influence on ROS level; At 24 h BAPTA-AM and NAC decreased the LDH level, EGTA had no influence on LDH level. Conclusion These data demonstrated that NaF can cause Ca2+ release from the site of [Ca²⁺]_i storage which causes ROS production in human neuroblastoma SH-SY5Y cells, and both of them induce cytotoxicity and the increase of LDH level.