Weiping Zhao

Exogenous melatonin modulates apoptosis in the mouse brain induced by high-LET carbon ion irradiation.

Abstract:  The aim of this study was to investigate whether melatonin, a free radical scavenger and a general antioxidant, regulates the brain cell apoptosis caused by carbon ions in mice at the level of signal transduction pathway. Young Kun‐Ming mice were divided into five groups: control group, irradiation group and three melatonin (1, 5, and 10 mg/kg daily for 5 days i.p.) plus irradiation‐treated groups. An acute study was carried out to determine oxidative status, apoptotic cells, and mitochondrial membrane potential (ΔΨm) as well as pro‐ and anti‐apoptotic protein levels in a mouse brain 12 hr after irradiation with a single dose of 4 Gy. In irradiated mice, a significant rise in oxidative stress and apoptosis (TUNEL positive) was accompanied by activated expression of Bax, cytochrome c, caspase‐3, and decreased ΔΨm level. Melatonin supplementation was better able to reduce irradiation‐induced oxidative damage marked by carbonyl or malondialdehyde content, and stimulate the antioxidant enzyme activities (superoxide dismutase and catalase) together with total antioxidant capacity. Moreover, administration with melatonin pronouncedly elevated the expression of Nrf2 which regulates redox balance and stress. Furthermore, melatonin treatment mitigated apoptotic rate, maintained ΔΨm, diminished cytochrome c release from mitochondria, down‐regulated Bax/Bcl‐2 ratio and caspase‐3 levels, and consequently inhibited the important steps of irradiation‐induced activation of mitochondrial pathway of apoptosis. Thus, we propose that the anti‐apoptotic action with the alterations in apoptosis regulator provided by melatonin may be responsible at least in part for its antioxidant effect by the abolishing of carbon ion–induced oxidative stress along with increasing Nrf2 expression and antioxidant enzyme activity.

Induction of cytogenetic adaptive response in spermatogonia and spermatocytes by pre-exposure of mouse testis to low-dose 12C6+ ions

To investigate the effects of pre-exposure of mouse testis to low-dose (12)C(6+) ions on cytogenetics of spermatogonia and spermatocytes induced by subsequent high-dose irradiation, the testes of outbred Kun-Ming strain mice were irradiated with 0.05Gy of (12)C(6+) ions as the pre-exposure dose, and then irradiated with 2Gy as challenging dose at 4h after per-exposure. Poly(ADP-ribose) polymerase (PARPs) activity and PARP-1 protein expression were respectively measured by using the enzymatic and Western blot assays at 4h after irradiation; chromosomal aberrations in spermatogonia and spermatocytes were analyzed by the air-drying method at 8h after irradiation. The results showed that there was a significant increase in the frequency of chromosomal aberrations and significant reductions of PARP activity and PARP-1 expression level in the mouse testes irradiated with 2Gy of (12)C(6+) ions. However, pre-exposure of mouse testes to a low dose of (12)C(6+) ions significantly increased PARPs activity and PARP-1 expression and alleviated the harmful effects induced by a subsequent high-dose irradiation. PARP activity inhibitor 3-aminobenzamide (3-AB) treatment blocked the effects of PARP-1 on cytogenetic adaptive response induced by low-dose (12)C(6+) ion irradiation. The data suggest that pre-exposure of testes to a low dose of heavy ions can induce cytogenetic adaptive response to subsequent high-dose irradiation. The increase of PARP-1 protein induced by the low-dose ionizing irradiation may be involved in the mechanism of these observations.

Results of carbon ion radiotherapy for skin carcinomas in 45 patients

Background  Heavy ions represent the best tool for external radiotherapy (RT) of inoperable tumours. Heavy ion RT has been used in the treatment of various tumours, especially for radioresistant tumours mediated by hypoxia, localized near organs at risk. Most of these treatments are concentrated in deep‐seated tumours such as those of the brain, head, lung, liver, rectum and urogenital organs, and treatment of skin carcinomas is limited.

Effects of carbon ion beam irradiation on lung injury and pulmonary fibrosis in mice

Radiation-induced lung injury is a well-described complication of nuclear accidents, marrow-transplant pretreatment and thoracic radiotherapy. The mechanism is complex and no special therapy for it is available at present. To study radiation pulmonary injury following heavy ion radiotherapy for thoracic tumors, Kunming mice were randomly divided into 4 groups: normal control and 2, 4 and 6 Gy irradiation groups which underwent whole-body exposure to 235 MeV/u 12C6+ administered at the Heavy Ion Research Facility in Lanzhou (HIRFL). The pathological changes were observed by hematoxylin and eosin staining and the hydroxyproline (HP) content was assessed by spectrophotometry at months 1, 2, 3, 4, 5 and 6 after radiation exposure. In addition, the expression of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β in the lung tissues was measured. The results showed that, compared with the control group, the lung tissue HP content was increased following irradiation but did not statistically significantly change after 4 months in the 4- and 6-Gy-treated groups. However, in the 2-Gy-treated group, the HP content was markedly increased between months 1 and 4 and decreased after month 4. The extent of the lung injury was significantly increased by the higher radiation dosages but was relieved in the 2 Gy group as the time since irradiation increased. The results also revealed that the levels of TNF-α were upregulated and reached a maximum at month 2, but decreased noticeably 2 months later in the experimental groups. The expression of TGF-β increased markedly in month 4 and was altered little in the 4- and 6-Gy-treated groups but decreased sharply in the 2 Gy irradiation group after month 4. These findings suggest that heavy ion radiotherapy for chest tumors causes lung injury to a certain extent, while there is likely to be little injury to lungs treated with <2 Gy, which provides scientific evidence for the use of heavy ion therapy for thoracic tumors.

NADPH Oxidase Activation Contributes to Heavy Ion Irradiation–Induced Cell Death

Increased oxidative stress plays an important role in heavy ion radiation–induced cell death. The mechanism involved in the generation of elevated reactive oxygen species (ROS) is not fully illustrated. Here we show that NADPH oxidase activation is closely related to heavy ion radiation–induced cell death via excessive ROS generation. Cell death and cellular ROS can be greatly reduced in irradiated cancer cells with the preincubation of diphenyleneiodium, an inhibitor of NADPH oxidase. Most of the NADPH oxidase (NOX) family proteins (NOX1, NOX2, NOX3, NOX4, and NOX5) showed increased expression after heavy ion irradiation. Meanwhile, the cytoplasmic subunit p47phox was translocated to the cell membrane and localized with NOX2 to form reactive NADPH oxidase. Our data suggest for the first time that ROS generation, as mediated by NADPH oxidase activation, could be an important contributor to heavy ion irradiation–induced cell death.

Potentiality of phosphorylation of BRCA1 at Ser 1524 to activate p21 in response to X-ray irradiation

The breast and ovarian cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein, which functions as a tumor suppressor gene. Many studies suggested that multiple functions of BRCA1 may contribute to its tumor suppressor activity, including roles in cell cycle checkpoints, apoptosis and transcription. It is postulated that phosphorylation of BRCA1 is an important means by which its cellular functions are regulated. In this study, we employed phospho-Ser-specific antibody recognizing Ser-1524 to study BRCA1 phosphorylation under conditions of DNA damage and the effects of phosphorylation on BRCA1 functions. The results showed that 10 Gy X-ray treatment significantly induced phosphorylation of Ser-1524 but not total BRCA1 protein levels. The expression both of p53 and p21 increased after irradiation, but ionizing radiation (IR) -induced activation of p21 was prior to that of p53. The percentages of G0/G1 phase remarkably increased after IR. In addition, no detectable levels of 89 kDa fragment of PARP, a marker of apoptotic cells, were observed. Data implied that IR-induced phosphorylation of BRCA1 at Ser-1524 might activatep21 protein, by which BRCA1 regulated cell cycle, but play no role in apoptosis.