The Study On HBCD Neural Developmental Toxicity Mechanism And HBCD Stereoselectivity And Enantioselectivity In Human Hepatic Cells Lines

HBCD belonges to low acute toxicity chemicals, but its endocrine disruption, immunotoxicity, reproduction toxicity, developmental toxicity and neural toxicity had been to well known for long years. Neural developmental toxicity was thought to be a sensitive endpoint for HBCD, oxidative stress followed by mitochondrial dysfunction which led to energy metabolism stress and mitochondrial dependent apoptosis was important mechanism for HBCD. Our research used human neuroblastoma cell line SH-SY5 Y as test model which induced differentiation by 2.5 ?g/m L ?-NGF to study neural developmental toxicity of HBCD, we detected the neurite outgrowth, apoptosis, ROS generation, mitochondrial membrane potential, intracellular calcium concentration, ATP production by techniques including flow cytometry, immunofluorescence chemistry, HPLC, q-PCR and western blot. The outcome showed us that:1. Differentiating neural cells were more vulnerable to HBCD than undifferentiated cells, HBCD caused higher rate of apoptosis in differentiating cells in a time dependent manner under the No Effect Concentration for undifferentiated cells. Cyt-c leakage from mitochondrial matrix, Apaf-1 and Caspase-9 expression increased showed a mitochondrial dependent apoptosis was triggered in the differentiating SH-SY5 Y.2. ROS generation, mitochondrial transmembrane potential rising and intracellular calcium mildly increasing were essential events for ?-NGF induced differentiation, but dramatically change of ROS generation, mitochondrial transmembrane potential break down and huge calcium interflow caused by HBCD would disturb the differentiation, antioxidant chemical NAC reduced ROS following mitochondrial transmembrane potential and intracellular calcium concentration recovered, and apoptosis rate decreased, which suggested the ROS leading mitochondrial dysfunction then triggered apoptosis.3. HBCD suppressed neurite outgrowth in SH-SH5 H induced by ?-NGF, MAP2, a neurite marker expression was down regulated followed with ATP and energy charge reducing, NAC alleviated the neurite outgrowth suppression, ATP production and cell energy charge increased, but MAP2 level did not change.4. The energy sensor AMPK was phosphorylated in a time-dependant manner in SH-SY5 Y when ?-NGF existed, which corresponding AMP/ATP changing, PI3K3 and AKT also appeared high level phosphorylation, while m TOR was down regulated; HBCD enhanced m TOR phosphorylation and suppressed AMPK phosphorylation, this phenomenon was inhibited by LY294002 or MK-2006(inhibitor for PI3 K and AKT), which following the increasing expression of MAP2. These results suggested HBCD down regulated AMPK phosphorylation through PI3K/AKT/m TOR pathway, which would be a reason for MAP2 expression inhibited by HBCD.?-, ?- and ?-HBCD are primary stereo isomers in industry HBCD, the proportion of ?-HBCD is about 75~89%, but in ambient medium or biota, this proportion varies largely from different samples, ?-HBCD were found to be the dominant stereoisomer in biota, its proportion reached 90% and more in high trophic level. The 3 stereo isomers have enantiomers individually,(+)-?-HBCD and(-)-?-HBCD,(+)-?-HBCD and(-)-?-HBCD,(+)-?-HBCD and(-)-?-HBCD. Except for stereoselectivity, enantioselectivity for HBCD in different environmental medium and different species is also reported. The stereoselectivity and enantioselectivity for HBCD vary in species and organs, there were no regular pattern to evaluate the isomers risk. Liver is the most important metabolite position for HBCD, this study used two hepatic cells, L-02 and Hep G2 to explored the HBCD stereoselectivity and enantioselectivity in human like tissue, the two cells lines were incubated with 10-7,10-6, 10-5 mol/L ?-??- and ?-HBCD respectively for 1 d?2 d?4 and 6 d, then LC-MS/MS was applied to separate the stereo isomers and enantiomers.,this study also tested 10-5 mol/L ?-??- and ?-HBCD toxicity in L-02 and Hep G2 cells. The results showed:1. ?-HBCD and ?-HBCD were isomerized to ?-HBCD, while ?-HBCD was not isomerized to ?-HBCD or ?-HBCD in Hep G2 cells, only little ?-HBCD was detected in ?-HBCD treated L-02 cells and no other isomerization product were found in L-02.2. The EF for ?-, ?-, ?-HBCD were 0.54±0.02, 0.80±0.02, 0.31±0.01 respectively in L-02 and 0.54±0.01, 0.79±0.02, 0.32±0.02 in Hep G2,(+)-?-HBCD and(-)-?-HBCD were dominant enantiomers in these two cells.3. ?-HBCD and ?-HBCD had more potent to suppress cell proliferation than ?-HBCD in L-02 cells, while in Hep G2 cells ?-HBCD showed stronger suppression effect than ?-HBCD and ?-HBCD; the ability to generate ROS and reducing mitochondrial transmembrane potential was ?-HBCD > ?-HBCD > ?-HBCD, and Hep G2 cells was more vulnerable than L-02 cells; the DNA damage test showed that ?-HBCD was the most effective DNA breaking stereo isomer, ?-HBCD was the weakest DNA breaker.4. CYP1A1 was down regulated by ?-HBCD in L-02 while ?-HBCD and ?-HBCD in Hep G2, CYP2B6 was up regulated by ?-HBCD and ?-HBCD in L-02,?-HBCD induced GST expression in L-02.Above all, we concluded that HBCD inhibited SH-SY5 Y differentiation through mitochondrial dependant apoptosis and neurite outgrowth suppressed by energy stress, PI3K/AKT/m TOR pathway played important roles in AMPK phosphorylation which adjusts ATP production and MAP2 expression.?-, ?- and ?-HBCD had stereoselectivity and enantioselectivity in L-02 and Hep G2 cells, the metabolite rate for the 3 stereo isomers is ?-HBCD > ?-HBCD > ?-HBCD, ?- and ?- HBCD were transformed to ?-HBCD in two cell lines,(+)-?-HBCD and (-)-?-HBCD were dominant enantiomers.The cellular toxicity for ?-, ?- and ?-HBCD was ?-HBCD > ?-HBCD > ?-HBCD, and Hep G2 was more sensitive to HBCD; while DNA damage potent was ?-HBCD > ?-HBCD > ?-HBCD and L-02 showed vulnerability; CYP1A1 was down regulated by ?-HBCD in L-02 and ?-HBCD, ?-HBCD in Hep G2, CYP2B6 was up regulated by ?-HBCD and ?-HBCD in L-02, and GST was up regulated by ?-HBCD, the difference between the P450 induce and GST expression in L-02 and Hep G2 perhaps were reasons for more toxicity by ?-HBCD and L-02 resistance to HBCD stereo isomers.