| Objective:This study was designed to study damage effects of benzene metabolites on bone marrow cell (BMC) of neonatal mice and mechanisms.Methods:Primary cultured bone marrow cell (BMC) of Kunming mice after ablactation for two weeks with body weight of 15±3g was used as model to exposed to 25~50μmol/L hydroquinone (HQ),catechol (CAT) and CAT+HQ.①MTT assay was performed to detect the inhibitory effect of HQ and CAT;②biochemical method to examine oxidative damage and activities of metabolic enzymes: myeloperoxidase (MPO) and glutathione S-transferase (GST);③flow cytometry (FCM) to detect changes of cell cycle and apoptosis; immunocytochemical method to observe expression of apoptotic related proteins in mitochondrial pathway;④3H-TdR incorporation assay to detect DNA damage; immunocytochemical method and Western Blot to examine expression of MPO;⑤R T-PCR to detect transcription level of GSTM1 gene. It was showed that HQ and CAT could inhibit growth of BMC of neonatal mice and their combination effects was synergistic; they also could suppress DNA synthesis and cause increase of unscheduled DNA synthesis; induce S phage arrest, cell apoptosis through mitochondrial pathway; induce oxidative damage; enhance the expression and activity of MPO and increase the transcription of GSTM1 and decrease activity of GST.Results:1. Toxic effect of benzene metabolite to primary cultured BMC of neonatal miceMTT assay was used to detect survival rate of BMC induced by CAT,HQ and HQ+CAT. After exposure to CAT for 12,24 and 48h, with the increase of CAT, survival rate of BMC at three time points decreased in a dose-dependent manner and statistically significant differences were found between exposure and NC groups at 12h in respect of changes in survival rate (P<0.05, P<0.01). After exposure to HQ for 12,24 and 48h, with the increase of HQ, survival rate of BMC at three time points decreased gradually compared with NC group (P<0.05, P<0.01). After exposure to HQ + CAT group for 12,24 and 48h, with the increase of dosage, survival rate of BMC at three time points decreased gradually in a dose-dependent manner compared with NC group (P<0.05, P<0.01). It was concluded that CAT and HQ had toxic effect on BMC of neonatal mice and their combination effects was synergistic.2. Oxidative damage of BMC of neonatal mice induced by benzene metaboliteBiochemical method was used to detect changes of SOD,GSH-PX activities and MDA content after exposure to 25,50,100μmol/L CAT,HQ and CAT+HQ groups for 24h. With the increase of exposure dosage, MDA content and SOD activity increased while GSH-Px activity decreased in all groups compared with NC group (P<0.01) . It indicated CAT,HQ and their joint action all could induce oxidative damage of BMC of neonatal mice. 3. Influences on total DNA synthesis and unscheduled DNA synthesis of BMC of neonatal mice induced by benzene metabolite3.1 Influences on total DNA synthesis of BMC of neonatal mice induced by benzene metaboliteRadioactive incorporation method was performed to detect total DNA synthesis of BMC after exposure to 25,50,100μmol/L CAT,HQ and CAT+HQ for 12,24 and 48h, respectively. With the increase of exposure dosage and period, cpm value in all groups decreased compared to NC group (P<0.01) .It indicated CAT,HQ and their joint action could inhibit DNA synthesis of BMC.3.2 Influences on unscheduled DNA synthesis of BMC of neonatal mice induced by benzene metaboliteHydrea was used to specifically block the DNA synthesis in S phage followed by 3H-TdR incorporation method to detect unscheduled DNA synthesis to observe DNA damage and repair. After exposure to 25,50,100μmol/L CAT for 12 and 24h, respectively. With the increase of exposure dosage and period, cpm value in all groups increased compared to NC group (P<0.01); while at 48h, the cmp value decreased. After exposure to 25,50,100μmol/L HQ for 12h. With the increase of exposure dosage, cpm value increased and there were significant differences of cmp between 50,100μmol/L groups and NC group (P<0.05,P<0.01), while after exposure for 24,48h, cmp value decreased. After exposure to 25,50,100μmol/L CAT+HQ for 12,24 and 48h, cmp value in all groups decreased. Therefore, it could be conclude that CAT,HQ and their joint action could influence unscheduled DNA synthesis of BMC of neonatal mice.4. Changes of MPO activity and expression of BMC of neonatal mice induced by benzene metabolite Biochemical method was used to detect changes of MPO activity. After exposure to 50μmol/L CAT,HQ and CAT+HQ groups for 24h, MPO activity in three groups ascended compared with NC group (P<0.001). Immunocytochemical method was used to detect changes of MPO expression. Except in 25μmol/L CAT groups, positive rate in all the other groups increased compared with NC group (P<0.01). Furthermore, with the method of Western Blot, it was showed expression of the 72kDa heavy chain of MPO and gray scale of MPO and GAPDH in all exposure groups was higher than NC group. It was demonstrated that CAT,HQ and their joint action could increased MPO activity and enhance the expression of MPO.5. Changes of GST activity and GSTM1 gene transcription of BMC of neonatal mice induced by benzene metaboliteBiochemical method was used to detect changes of GST activity. After exposure to 50μmol/L CAT,HQ and CAT+HQ groups for 24h, total GST activities in three groups showed a decreased tendency compared with NC group (P<0.01). Meanwhile, RT-PCR was applied to examine mRNA level of GSTM1 and it was observed that mRNA level in three groups was higher than NC group. Therefore, it could be concluded that CAT,HQ and their joint action could increase mRNA level of GSTM1 of BMC of neonatal mice but total GST activities still appeared a descending tendency.6. Changes of cell cycle of primary cultured BMC of neonatal mice induced by benzene metaboliteFCM together with PI dying methods was performed to examine changes of cell cycle of BMC of neonatal mice. After exposure to 50μmol/L CAT,HQ and CAT+HQ groups for 12,24 and 48h respectively, except for CAT group at 12h, with the increase of exposure period, cells in S phage at different exposure groups increased compared with NC group (P<0.05, P<0.01). While 12 and 24h after exposed to three groups, cell s in G2+M phage all decreased compared with NC group (P<0.05, P<0.01). It was showed CAT,HQ and CAT+HQ could cause cell increase in S phage while decrease in G2+M leading to the occurrence of S phage arrest with a time-effect relationship and CAT+HQ appeared stronger effect.7. BMC apoptosis of neonatal mice induced by benzene metabolite7.1 Apoptotic rate of neonatal mice induced by benzene metaboliteFCM together with PI dying methods was performed to examine apoptosis. After exposure to 50μmol/L CAT,HQ and CAT+HQ groups for 12,24 and 48h respectively, except HQ groups, BMC apoptotic rate in other groups increased with the prolonged exposure period compared to NC group (P<0.01) indicating benzene metabolite could induce apoptosis of BMC with a time-effect relationship.7.2 Expression of apoptosis related proteins in mitochondrial pathway induced by benzene metaboliteImmuocytochemical method was applied to detect changes of expression of Cyt-c,Bax,Bcl-2 and Caspase-3 in BMC of neonatal mice after exposure to 25,50,100μmol/L CAT,HQ and CAT+HQ groups for 12h. Except in 25μmol/L CAT and 25μmol/L HQ groups, expressions of Cyt-c,Bax and Caspase-3 in other groups enhanced with the increase of dosage compared to NC group (P<0.05, P<0.01) and there also was significant difference in expression between CAT+HQ group and CAT or HQ groups; Moreover, expression of Bcl-2 in all exposure groups weakened with the increase of dosage compared to NC group (P<0.01) and there also was significant difference in expression between CAT+HQ group and CAT or HQ groups. It was indicated that mitochondrial pathway probably involved in the apoptosis of BMC of neonatal mice induced by CAT and HQ. Conclusions:1.After exposure to 25,50,100μmol/L CAT,HQ and CAT+HQ for 12,24 and 48h, growth of BMC was inhibited and the joint action showed a kind of synergistic effect.2. After exposure to CAT,HQ and CAT+HQ for 24h, SOD activity increased while GSH-Px decreased and MDA content increased leading to oxidative damage of BMC of neonatal mice.3.CAT,HQ and their combination could inhibit total DNA synthesis of BMC of neonatal mice .4. CAT,HQ and their combination could influence unscheduled DNA synthesis of BMC of neonatal mice.5.CAT,HQ and their combination could increase activity and expression of MPO.6. CAT,HQ and their combination could enhance mRNA level of GSTM1 of BMC of neonatal mice but total GST activities still appeared a descending tendency.7. CAT,HQ and CAT+HQ could cause cell increase in S phage while decrease in G2+M leading to the occurrence of S phage arrest with a time-effect relationship and CAT+HQ appeared stronger effect.8. CAT,HQ and CAT+HQ could induce apoptosis of BMC with a time- effect relationship, their combination showed stronger effect.9. CAT,HQ and CAT+HQ could increase activity and expression of Cyt-c,Bax and Caspase-3, decrease activity and expression of Bcl-2.10. Mitochondrial pathway probably involved in the apoptosis of BMC of neonatal mice induced by CAT,HQ and their combination.
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