Mitochondrial modulation of apoptosis induced by low-dose radiation in mouse testicular cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate whether apoptosis induced by low-dose radiation (LDR) is regulated by mitochondrial pathways in testicular cells.</p><p><b>METHODS</b>Male mice were exposed to whole-body LDR, and changes in mitochondrial function and in expression of apoptotic factors were analyzed in the testicular cells as follows. Total nitric-oxide synthase (T-NOS) and Na+/K+ ATPase activities were biochemically assayed. Reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) were determined by flow cytometry using fluorescent probes. Levels of mRNAs encoding cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) were quantified by real-time reverse-transcription PCR (RT-PCR). Expression of Cyt c, AIF, caspase-9, and caspase-3 at the protein level was assessed by western blotting and immunohistochemistry.</p><p><b>RESULTS</b>LDR induced an increase in T-NOS activity and ROS levels, and a decrease in Na+/K+ ATPase activity and mitochondrial Δψm, in the testicular cells. The intensity of these effects increased with time after irradiation and with dose. The cells showed remarkable swelling and vacuolization of mitochondria, and displayed a time- and dose-dependent increase in the expression of Cyt c, AIF, procaspase-9, and procaspase-3. Activation of the two procaspases was confirmed by detection of the cleaved caspases. The changes in expression of the four apoptotic factors were mostly limited to spermatogonia and spermatocytes.</p><p><b>CONCLUSION</b>LDR can induce testicular cell apoptosis through mitochondrial signaling pathways.</p>

Involvement of endoplasmic reticulum stress in apoptosis of testicular cells induced by low-dose radiation / 华中科技大学学报（医学）（英德文版）

The study examined the role of endoplasmic reticulum stress (ERS) and signaling pathways of inositol-requiring enzyme-1 (IRE1), RNA-activated protein kinase-like ER kinase (PERK) and activating transcription factor-6 (ATF6) in apoptosis of mouse testicular cells treated with low-dose radiation (LDR). In the dose-dependent experiment, the mice were treated with whole-body X-ray irradiation at different doses (25, 50, 75, 100 or 200 mGy) and sacrificed 12 h later. In the time-dependent experiment, the mice were exposed to 75 mGy X-ray irradiation and killed at different time points (3, 6, 12, 18 or 24 h). Testicular cells were harvested for experiments. H(2)O(2) and NO concentrations, and Ca(2+)-ATPase activity were detected by biochemical assays, the calcium ion concentration ([Ca(2+)]i) by flow cytometry using fluo-3 probe, and GRP78 mRNA and protein expressions by quantitative real-time RT-PCR (qRT-PCR) and Western blotting, respectively. The mRNA expressions of S-XBP1, JNK, caspase-12 and CHOP were measured by qRT-PCR, and the protein expressions of IRE1α, S-XBP1, p-PERK, p-eIF2α, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP by Western blotting. The results showed that the concentrations of H2O2 and NO, the mRNA expressions of GRP78, S-XBP1, JNK, caspase-12 and CHOP, and the protein expressions of GRP78, S-XBP1, IRE1α, p-PERK, p-eIF2α, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP were significantly increased in a time- and dose-dependent manner after LDR. But the [Ca(2+)]i and Ca(2+)-ATPase activities were significantly decreased in a time- and dose-dependent manner. It was concluded that the ERS, regulated by IRE1, PERK and ATF6 pathways, is involved in the apoptosis of testicular cells in LDR mice, which is associated with ERS-apoptotic signaling molecules of JNK, caspase-12 and CHOP.

Involvement of endoplasmic reticulum stress in apoptosis of testicular cells induced by low-dose radiation / 华中科技大学学报（医学）（英德文版）

The study examined the role of endoplasmic reticulum stress (ERS) and signaling pathways of inositol-requiring enzyme-1 (IRE1), RNA-activated protein kinase-like ER kinase (PERK) and activating transcription factor-6 (ATF6) in apoptosis of mouse testicular cells treated with low-dose radiation (LDR). In the dose-dependent experiment, the mice were treated with whole-body X-ray irradiation at different doses (25, 50, 75, 100 or 200 mGy) and sacrificed 12 h later. In the time-dependent experiment, the mice were exposed to 75 mGy X-ray irradiation and killed at different time points (3, 6, 12, 18 or 24 h). Testicular cells were harvested for experiments. H2O2 and NO concentrations, and Ca(2+)-ATPase activity were detected by biochemical assays, the calcium ion concentration ([Ca(2+)]i) by flow cytometry using fluo-3 probe, and GRP78 mRNA and protein expressions by quantitative real-time RT-PCR (qRT-PCR) and Western blotting, respectively. The mRNA expressions of S-XBP1, JNK, caspase-12 and CHOP were measured by qRT-PCR, and the protein expressions of IRE1α, S-XBP1, p-PERK, p-eIF2α, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP by Western blotting. The results showed that the concentrations of H2O2 and NO, the mRNA expressions of GRP78, S-XBP1, JNK, caspase-12 and CHOP, and the protein expressions of GRP78, S-XBP1, IRE1α, p-PERK, p-eIF2α, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP were significantly increased in a time- and dose-dependent manner after LDR. But the [Ca(2+)]i and Ca(2+)-ATPase activities were significantly decreased in a time- and dose-dependent manner. It was concluded that the ERS, regulated by IRE1, PERK and ATF6 pathways, is involved in the apoptosis of testicular cells in LDR mice, which is associated with ERS-apoptotic signaling molecules of JNK, caspase-12 and CHOP.

Low-dose radiation induces endoplasmic reticulum stress and activates PERK-CHOP signaling pathway in mouse testicular cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the correlation of low-dose radiation with endoplasmic reticulum stress and the activation of the PERK-CHOP signaling pathway in mouse testicular cells.</p><p><b>METHODS</b>Healthy Kunming mice were randomly assigned to time-effect (0, 3, 6, 12 and 24 h of irradiation at 75 mGy) and dose-effect (12 h of irradiation at 0, 50, 75, 100 and 200 mGy) groups. The contents of H202 and MDA were measured by colorimetry with the agent kits, the expressions of GRP78, PERK and CHOP mRNA detected by quantitative RT-PCR, and the levels of GRP7B, PERK, phosphorylated PERK (pho-PERK) and CHOP proteins determined by Western blotting and image analysis.</p><p><b>RESULTS</b>After whole-body irradiation of the mice with 75 mGy, the content of H2 02 in the testis tissue was increased with time prolongation, while that of MDA decreased slightly at 3 and 6 h and then increased with the lengthening of time, both increased significantly at 12 and 24 h as compared with those at 0 h (P < 0. 05, P < 0. 01). Apart from reduced levels of GRP78 mRNA at 3 and 24 h and GRP78 protein at 6 h after irradiation, significant increases were found in the mRNA expressions of GRP78 at 12 h, PERK at 3,6, 12 and 24 hand CHOP at 12 and 24 h (P < 0.05, P < 0.01), as well as in the protein levels of GRP78 at 12 and 24 h, pho-PERK at 3, 12 and 24 h and CHOP at 3, 6, 12 and 24 h in comparison with those at 0 h (P < 0. 05, P < 0. 01). No obvious regularity was observed in the change of the PERK protein expression. After 12 h of whole-body irradiation, the content of H202 was increased at 50, 75 and 100 mGy, but decreased slightly at 200 mGy, while that of MDA was increased with dose increasing, with significant increases in the content of H2 02 at 75 and 100 mCy and in that of MDA at 75, 100 and 200 mGy as compared with the 0 mGy group. Apart from the reduced levels of GRP78 mRNA at 50 and 200 mCy, significant increases were found in the mRNA expressions of PERK at 75, 100 and 200 mGy and CHOP at 50, 75, 100 and 200 (P c 0. 05, P < 0.01) as well as in the protein levels of GRP78 at 100 and 200 mGy, pho-PERK at 50, 100 and 200 mGy and CHOP at 50, 75, 100 and 200 mCy as compared with those at 0 mGy (P < 0. 05, P < 0. 01). There were differences in the changes of different protein expressions, but no obvious regularity was seen in the change of the PERK protein expression.</p><p><b>CONCLUSION</b>Low-dose radiation can induce endoplasmic reticulum stress in mouse testicular cells, and activate the PERK-CHOP signaling pathway.</p>

Increased levels of p53 and PARP-1 in EL-4 cells probably related with the immune adaptive response induced by low dose ionizing radiation in vitro / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>This paper is to explore the DNA repair mechanism of immune adaptive response (AR) induced by low dose radiation (LDR), the changes of mRNA levels and protein expressions of p53, ATM, DNA-PK catalytic subunit (DNA-PKcs) and PARP-1 genes in the LDR-induced AR in EL-4 cells.</p><p><b>METHODS</b>The apoptosis and cell cycle progression of EL-4 cells were detected by flow cytometry in 12 h after the cells received the pre-exposure of 0.075 Gy X-rays (inductive dose, D1) and the succeeding high dose irradiation (challenge dose, D2; 1.0, 1.5, and 2.0 Gy X-rays, respectively) with or without wortmannin (inhibitor of ATM and DNA-PK) and 3-aminobenzamid (inhibitor of PARP-1). And the protein expressions and mRNA levels related to these genes were detected with flow cytometry and reverse transcription-polymerase chain reaction in 12 h after irradiation with D2.</p><p><b>RESULTS</b>The mRNA and protein expressions of p53 and PARP-1 in EL-4 cells in the D1 + D2 groups were much lower than those in the D2 groups, and those of PARP-1 in the 3-AB + D2 and the 3-AB + D1 + D2 groups were much lower than those in the D2 and the D1 + D2 groups. The percentage of apoptotic EL-4 cells in the 3-AB + D1 + D2 groups was much higher than that in the D1 + D2 groups, that in the G₀/G₁ and the G₂ + M phases was much higher, and that in the S phase were much lower. Although the ATM and DNA-PKcs mRNA and protein expressions in wortmannin + D1 + D2 groups were much lower than those in the D1 + D2 groups, there were no significant changes in the apoptosis and cell cycle progression between the wortmannin + D1 + D2 and the D1 + D2 groups.</p><p><b>CONCLUSION</b>PARP-1 and p53 might play important roles in AR induced by LDR.</p>

Effects of corticosterone, cAMP, cGMP, Ca2+, and protein kinase C on apoptosis of mouse thymocytes induced by X-ray irradiation / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To observe the effects of signal factors of corticosterone (CS), cAMP, cGMP, Ca2+ andprotein kinase C (PKC) on lymphocyte apoptosis in mouse thymus induced by X-rays of 4 Gy in vitro.</p><p><b>METHODS</b>The DNA lytic rate for thymocytes was measured by fluorospectrophotometry.</p><p><b>RESULTS</b>The DNA lyric rate for thymocytes 4-8 hours after irradiation with 2-8 Gy was significantly higher than that in the control (P<0.01). As compared with the control, the DNA lytic rate for thymocytes treated with 0.01 micromol/L CS (P<0.01), 50 ng/mL cAMP (P<0.01), 0.05-0.4 microg/mL ionomycin (Iono, P<0.05 or P<0.01) or 0.05-0.4 ng/mL phorbol myristate acetate (PMA, P<0.05 or P<0.01), respectively, was significantly increased, while the rate for thymocytes treated with 50 ng/mL cGMP was not significantly increased. The DNA lytic rate for thymocytes treated with 0.01 micromol/L CS (P<0.01), 50 ng/mL cAMP (P<0.01), 0.2 and 0.4 microg/mL Iono (P<0.05), and 0.2 and 0.4 ng/mL PMA (P<0.05) plus 4-Gy irradiation, respectively, was significantly higher than that treated with single 4-Gy irradiation, while the rate for thymocytes treated with 50 ng/mL cGMP plus 4-Gy irradiation was not increased. When both 0.4 microg/mL Iono and 0.4 ng/mL PMA acted on the thymocytes, the DNA lytic rate for thymocytes was significantly higher than that in the control (P<0.01), the DNA lytic rate for thymocytes treated with both 0.4 microg/mL Iono and 0.4 ng/mL PMA plus 4-Gy irradiation was significantly higher than that treated with single 4-Gy irradiation (P<0.05), but was not significantly higher than that treated with 0.4 microg/mL Iono plus 4-Gy irradiation or 0.4 ng/mL PMA plus 4-Gy irradiation.</p><p><b>CONCLUSION</b>CS, cAMP, Ca2+, and PKC signal factors can promote thymocyte apoptosis induced by larger dose X-rays.</p>

Changes of cycle and apoptosis of spermatogenic cells and antioxidant capacity in male rats with diabetes mellitus / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the changes of cycle and apoptosis of spermatogenic cells and antioxidant capacity of the serum and testis in male rats with diabetes mellitus.</p><p><b>METHODS</b>Thirty male rats were divided into two groups, 10 for normal control and 20 for the diabetes group. The rats were injected intraperitoneally with streptozocin (TZ) to develop diabetes, and 12 weeks later, their survival rate and testis weight were recorded. The percentage of G0/G, S and G2/M phases and apoptosis in spermatogenic cells were measured with flow cytometry (FCM). Malondialdehyde (MDA) and nitric oxide (NO) levels, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and NO synthase (NOS) activities in the serum and testis were measured with thiobarbituric acid reactive substances (TBARs), nitric acid reoxidized enzyme, xanthine oxidative enzyme, 5,5 Dithiobis (2,2 nitrobenzoate) (TNB) and visible light photometer methods, respectively.</p><p><b>RESULTS</b>Twelve weeks after the male rats got diabetes, their survival rate, body weight and testis weight were significantly lower (p < 0.05), and the percentages of G0/G1 phases and apoptotic spermatogenic cells were obviously higher (P < 0.05) than the normal control. At the same time, the percentage of S and G2/M phases spermatogenic cells decreased. So the spermatogenic cells were arrested in G0/G1 phase. In the diabetic rat serum and testis, especially in the testis, MDA levels were distinctly higher and SOD activities were significantly lower than those in the control. Serum GSH-Px activities of the diabetic rats were significantly lower (p < 0.05), while testis GSH-Px activities were significantly higher than those in control group (P < 0.01). NO contents in the serum and testis of the diabetic rats (P < 0.01) increased significantly, particularly the former, while NOS activities in the serum decreased significantly as compared with the control (P < 0.5).</p><p><b>CONCLUSION</b>The increase in testis and serum MDA levels and NO contents and the decrease in the antioxidant enzyme activity of the diabetic rats may be relevant to spermatogenic disorder caused by the increase of G0/G1 phases arrest and spermatogenic cells apoptosis.</p>