芥子气体内生物标志物的检测及其代谢与分布研究进展

芥子气（sulfurmustard,HD）是一种糜烂性化学战剂,能造成皮肤、眼及呼吸系统等的损伤。虽然芥子气作用机制的研究开展较早,但迄今尚未完全阐明。芥子气中毒后可产生多种生物标志物,主要包括水解、氧化产物,谷胱甘肽、蛋白及DNA加合物,本文重点围绕这些生物标志物的检测技术、体内分布及代谢行为等研究进展进行评述。     

抑制DNA合成药物作用方式的简易测定法

很多药物(特别是抗癌药物)是通过抑制靶细胞内DNA的生物合成而发挥疗效的。为了阐明这一类型药物作用的分子机制,首先应区分作用方式是属于损伤DNA模板型还是干扰代谢型。一般说来,DNA的损伤作用近乎不可逆;干扰代谢的作用绝大多数具有可逆性,一旦药物作用被解除,细胞内DNA合成即可逐渐复原。根据这一原理,可利用肿瘤细胞体外解除药物作用后~3H-TdR掺入(代表DNA合成)的图谱(以下称掺入曲线)来测定药物的作用方式。     

内质网应激与肿瘤细胞耐药的相关机制

内质网是真核细胞内一种重要的细胞器,当细胞低氧、糖类供应不足或有化学药物处理时均可引发内质网应激(endoplasmic reticulum stress,ERS)。目前研究表明,内质网应激可激活多条通路,其中以未折叠蛋白反应(unfolded protein response,UPR)通路研究最为广泛。UPR通路激活后,可参与调控肿瘤细胞耐药。其中,参与的机制涉及DNA损伤修复、凋亡抑制、自噬等。     

过亚硝酸根对DNA损伤的研究进展

过亚硝酸根(ONOO )产生于一氧化氮自由基与超氧阴离子的反应。过亚硝酸根具有强细胞毒性，能够修饰DNA碱基、造成DNA单链断裂。DNA损伤会对生物体产生一系列变化如生物酶的激活、细胞凋亡等。这些变化与人类疾病相关。近年来过亚硝酸根对DNA损伤的研究倍受关注。综述过亚硝酸根对DNA的损伤形式、作用机制、病理现象的研究进展。     

新型PARP抑制剂veliparib

肿瘤细胞能够激活自身DNA的损伤修复机制进行修复,从而导致其对抗肿瘤药物和放疗产生耐药性,而聚腺苷酸二磷酸核糖转移酶(poly ADP-ribose polymerase,PARP)是一种DNA修复酶,在DNA修复通路中起关键作用.veliparib 是一种新型高选择抑制PARP的苯并咪唑类化合物,体内外实验表明本品具有显著的抑制PARP活性的作用.在治疗转移性乳腺癌、结肠癌、转移性黑色素瘤和脑肿瘤方面已取得显著的效果,其与替莫唑胺联用治疗乳腺癌的研究即将进入Ⅲ期临床.     

芥子气中毒机制及其防治药物研究

芥子气(mustard gas)是一种糜烂性毒剂，由于其难防难治，至今仍为外国军队化学仪器库的制式装备。近年来，国内外对芥子气全身吸收中毒的机制进行了广泛而深入的研究，并从中发现了疗效较好的防治药物。我们就几类典型的中毒机制学说及其代表药物综述如下。     

芥子气诱导大鼠小肠上皮细胞的凋亡

芥子气是一种双功能烃化剂,易与细胞内ＤＮＡ、ＲＮＡ和蛋白质等大分子物质起烃化反应,中毒后可以引起机体组织细胞的损伤以至死亡。细胞死亡方式有凋亡和坏死两种,但芥子气引起细胞坏死方式国内外还很少报道。小肠粘膜分裂旺盛的上皮细胞对芥子气较为敏感,致死剂量芥子气中毒动物的死亡高峰出现在中毒后第４,５ｄ,其主要原因是由于肠道严重失水所导致的低血容量性休克所致。本实验以大鼠小肠上皮细胞（ＩＥＣ６）为体外实验细胞模型,研究不同浓度芥子气对ＩＥＣ６细胞生长的影响及其引起的细胞死亡的方式。采用胎盘蓝染色法,发…     

百草枯中毒机制及相关药物治疗进展

目的:为临床抢救百草枯中毒提供新的药物,以延长患者生命,降低死亡率。方法:查阅国内、外相关文献,综述百草枯毒动学、中毒机制以及相关治疗。结果与结论:针对抗氧自由基、抗免疫、抗线粒体损伤治疗百草枯中毒的药物较成熟。而针对抗核因子-кB(NF-кB)的激活、抗脱氧核糖核酸(DNA)的氧化性损伤的治疗药物较少,因此此类药物有待进一步开发。     

芥子气眼部损伤研究进展

<正>芥子气是一种亲脂和烷化的化学糜烂性毒剂,以液滴态、雾态、蒸汽态长期储存。战争状态可通过多途径中毒,穿透能力强,病程长,易导致大量战斗减员和长期伤残。据资料记载,两伊战争中芥子气眼部损伤率高达77.61%～92.6%本文就芥子气体内代谢与毒理、眼部损伤的特征及角膜损伤的研究现状作一综述。1芥子气体内代谢与毒理作用     

抗氧化天然产物对芥子气氧化损伤的保护作用

目的 观察丹参、知母提取物对芥子气中毒所致氧化损伤的影响,评价其对芥子气中毒的预防和(或)治疗效果。方法 (1) 细胞水平试验：乳腺癌细胞MDA-MB-231和血管平滑肌细胞(VSMC)与芥子气200 μmol·L^-1作用15 min;受试药为丹酚酸4 mg、知母黄酮16 mg（1∶4）或丹酚酸4 mg、知母黄酮8 mg（1∶2）溶于50 μl DMSO,无血清DMEM稀释至100和50 mg·L^-1。预防给药时,药物与细胞预孵2 h,然后染毒; 治疗用药时,细胞染毒后与药物孵育24 h。分别观察细胞存活率并检测MDA水平。(2) 整体动物试验：雄性SD大鼠随机分为正常对照组、中毒对照组、维生素C（200 mg·kg^-1）治疗组（阳性对照）、抗氧化剂低剂量治疗组、中剂量治疗和高剂量治疗组。芥子气染毒剂量为3.5 mg·kg^-1（70%LD₅₀）,皮下注射,1 h后给予治疗;按丹酚酸∶知母黄酮1∶4,每日ig给予30, 60和120 mg·kg^-1,连续7 d。观察大鼠生存状况,测定血清中MDA, GSH和SOD水平,并取肠、肝进行病理学检查。结果 不同浓度的提取物复方对芥子气细胞毒性均无预防作用,但有一定治疗作用;复方提取物对芥子气引起的细胞氧化损伤有一定防护效果。提取物复方可提高芥子气中毒动物存活率;对脂质氧化和SOD抗氧化体系无明显影响;但可剂量依赖性地恢复芥子气中毒后的GSH水平。结论 芥子气对SD大鼠引起的氧化损伤可能主要由GSH体系介导,丹参、知母提取物通过升高GSH水平,发挥抗氧化作用,进而起到抗芥子气中毒效果。     

A dorsal model for cutaneous vesicant injury by 2-chloroethyl ethyl sulfide using C57BL/6 mice

To evaluate stem cell-derived therapeutics for cutaneous vesicant injuries, we developed a dorsal exposure model using C57BL/6 black mice and half-mustard, 2-chloroethyl ethyl sulfide (CEES). The dorsal side of a mouse was exposed to 1-5 microl of CEES for 10 minutes and then decontaminated. The data demonstrate that 3 microl of CEES induced edema and erythema that peaked 24 h post exposure. Histopathology showed a central area of deep injury characterized by severe necrosis of epidermis and dermis. The C57BL/6 is a unique model that can be used to unravel the molecular mechanism of injury, identify the effects of black skin pigmentation, and evaluate the efficacy of stem cell therapeutics for cutaneous vesicant exposure.     

Comparison of injuring effects of vesicant, irritant, and nonvesicant anticancer drugs on endothelial cells

Anticancer drugs are classified as vesicant, irritant, and nonvesicant drugs on the basis of frequency of their vascular disorder. In this study, we compared the injuring effects of three typical anticancer drugs of each class on porcine aorta endothelial cells (PAECs). The concentration inducing 50% cell viability inhibition was lower in the order of vesicant, irritant, and nonvesicant drugs. These results suggest that injuring effects of anticancer drugs on PAECs may be relevant as an indicator of frequency of their vascular disorder, and that this experimental model may be useful for the study of vascular disorder.     

Mechanism of DNA damage and apoptosis induced by anticancer drugs through generation of reactive oxygen species

A number of anticancer drugs exert their effect by causing DNA damage and subsequent apoptosis induction. Reactive oxygen species (ROS), such as hydrogen peroxide (H(2)O(2)) and super oxide anion (O(2)(-)), participate in apoptosis and DNA damage induced by some anticancer drugs, however, the precise mechanism of apoptosis via ROS formation remains to be clarified. I investigated the mechanism of apoptosis and DNA damage induced by anticancer drugs, especially topoisomerase inhibitors, using human cultured cells. TAS-103, a topoisomerase inhibitor, induces apoptosis through DNA cleavage and subsequent H(2)O(2) generation mediated by poly (ADP-ribose) polymerase (PARP) and NAD(P)H oxidase activation. Doxorubicin (DOX), an anthracycline antibiotic and topoisomerase inhibitor, induces apoptosis through direct oxidative DNA damage leading to indirect H(2)O(2) generation mediated by PARP and NAD(P)H oxidase activation. DOX caused site-specific oxidative DNA damage in the presence of copper(II), which may contribute to apoptosis. These findings suggest that ROS formation plays important roles in apoptosis induced by anticancer drugs. Furthermore, these studies may provide an insight into the development of new effective chemotherapeutic drugs.     

Ocular injuries following sulfur mustard exposure—Pathological mechanism and potential therapy

Sulfur mustard (SM) is a potent vesicant, known for its ability to cause incapacitation and prolonged injuries to the eyes, skin and respiratory system. The toxic ocular events following sulfur mustard exposure are characterized by several stages: photophobia starting a few hours after exposure, an acute injury phase characterized by inflammation of the anterior segment and corneal erosions and a delayed phase appearing following a clinically silent period (years in human). The late injury appeared in part of the exposed eyes, expressed by epithelial defects and corneal neovascularization (NV), that lead to vision deficits and even blindness. During the last years we have characterized the temporal development of ocular lesions following SM vapor exposure in rabbits and have shown the existence of two sub-populations of corneas, those exhibiting delayed ocular lesions (clinically impaired) and those exhibiting only minor injuries if at all (clinically non-impaired). The aim of the present study was to investigate the pathological mechanism underlying the delayed injury by focusing on the unique characteristics of each sub-population and to test the efficacy of potential treatments.     

Potential anti-inflammatory treatments against cutaneous sulfur mustard injury using the mouse ear vesicant model

In spite of several decades of research, no effective treatment to skin injuries following exposure to sulfur mustard (HD) has yet been found. In the present study, the mouse ear vesicant model was applied to awake mice in order to evaluate the efficiency of potential anti-inflammatory treatments in preventing HD-induced skin damages. Clinical follow-up and histological evaluation were used to characterize the injuries to the skin and to evaluate the efficiency of the drugs that were applied. Thus, the extent of mouse ear oedema and the histopathological changes following a single application of 0.2 or 1 microL of neat HD for 10 min (representing moderate and severe lesions, respectively), were monitored. Typical HD skin lesions were observed including epithelial and dermal damage. The development of the injury in mouse ears was found to be very similar to that reported in human skin. Screening of post-exposure topical steroids and non-steroidal antiinflammatory drugs (NSAIDs) proved that HD-induced inflammation could be diminished significantly as long as the treatment was applied during the early stages following exposure. A combined application of these drugs approved to be particularly effective in reducing inflammation.     

Response of mouse brain to a single subcutaneous injection of the monofunctional sulfur mustard, butyl 2-chloroethyl sulfide (BCS)*

Exposure to mustard-type vesicants results in alkylation of DNA and vesication. However, the biochemical mechanism for vesicant injury and whether it is localized or diffuse are not clear. We postulated that vesicant damage is mediated by free radicals, resulting in oxidative stress. These free radicals-mediated reactions may propagate systemically distal to the site of exposure. To test this hypothesis, we examined the effects of a single subcutaneous injection of the monofunctional sulfur mustard, butyl 2-chloroethyl sulfide (BCS), on the brain. We injected 3 groups (6 mice/group) of 5-month-old male, athymic, nude mice, weighing 30-35 g, subcutaneously with neat (undiluted) BCS (5 microliters/mouse). After 1, 24, and 48 h, we sacrificed the treated mice along with an untreated control group and analyzed the brains for biochemical markers of oxidative stress. Compared to untreated controls, the activity of glutathione peroxidase increased by 76%, P less than 0.005 at 24 h, and that of glutathione S-transferases by 25-37%, P less than 0.05 over the entire period. Total glutathione content in the brain was significantly lower, 17%, after 1 h and 23% after 24 h. We found also, concomitant with decreased glutathione, almost a 3-fold increase in susceptibility to lipid peroxidation. Because these changes are consistent with oxidative stress, we conclude that the effect of BCS administered subcutaneously may be translocated, reaching mouse brain, and causing oxidative stress.     

Delayed apoptosis and its regulation in astrocytes

Astrocytes, the most abundant glial cell type in the brain, are considered to have physiological and pathological roles in neuronal activities. We found that reperfusion of cultured astrocytes after Ca2+ depletion causes Ca2+ overload followed by delayed cell death and the Na(+)-Ca2+ exchanger in the reverse mode is responsible for this Ca(2+)-mediated cell injury (Ca2+ paradox injury). The Ca2+ paradox injury of cultured astrocytes is considered to be an in vitro model of ischemia/reperfusion injury, since a similar paradoxical change in extracellular Ca2+ concentration is reported in ischemic brain tissue. This review summarizes the mechanisms underlying the Ca(2+)-mediated injury of astrocytes and the protective effects of drugs against Ca2+ reperfusion injury. This study shows that Ca2+ reperfusion injury of astrocytes is accompanied by apoptosis as evidenced by DNA fragmentation and nuclear condensation. Calpain, reactive oxygen species, calcineurin, caspase-3, and NF-kappa B are involved in Ca2+ reperfusion-induced delayed apoptosis of astrocytes. Several drugs including CV-2619, T-588 and ibudilast protect astrocytes against the delayed apoptosis. CV-2619 prevents astrocytes from the delayed apoptosis by production of nerve growth factor, resulting in an activation of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 (PI3) kinase signal pathways. The protective effect of T-588 is mainly mediated by an activation of MAP/ERK signal cascade. Moreover, ibudilast prevents the Ca2+ reperfusion-induced delayed apoptosis of astrocytes via cyclic GMP signaling pathway. Further studies in this system will contribute to the development of new drugs that attenuate ischemia/reperfusion injury via modulation of astrocytes.     

Low-dose sulfur mustard primes oxidative function and induces apoptosis in human polymorphonuclear leukocytes

Although considerable work has focused on understanding the processes of direct tissue injury mediated by the chemical warfare vesicant, sulfur mustard (2,2'-bis-chloroethyl sulfide; SM), relatively little is known regarding the mechanisms of secondary injury caused potentially by the acute inflammatory response that follows SM exposure. Polymorphonuclear leukocytes (PMNs) play a central role in the initiation and propagation of inflammatory responses that, in some cases, result in autoimmune tissue damage. The potential for PMN-derived tissue damage following SM exposure may, in part, account for the protracted progression of the injury before it resolves. The current study was undertaken to evaluate the priming, oxidative function, and viability of PMN following exposure to low doses of SM such as those that might remain in tissues as a result of topical exposure. Our results demonstrate that doses of SM ranging from 25 to 100 microM primed PMN for oxidative burst in response to activation by fMLP, and that doses of SM ranging from 50 to 100 microM induced PMN apoptosis. Understanding the mechanisms through which SM directly affects PMN activation and apoptosis will be of critical value in developing novel treatments for inflammatory tissue injury following SM exposure.     

Amelioration of sulfur mustard skin injury following a topical treatment with a mixture of a steroid and a NSAID

The ability to ameliorate sulfur mustard (HD)-induced oedema by treatment with anti-inflammatory drugs was reported previously after screening four steroids and four non-steroidal anti-inflammatory drugs (NSAIDs) using the mouse ear vesicant model. Following the screening study, one steroid and one NSAID (Adexone and Voltaren) were selected as the most effective, and a mixture of the two was chosen for the present more extensive research. The effect of the combined treatment on clinical, biochemical and histopathological parameters following HD insult was studied. Mice ears were exposed to 0.2 micro l of HD for 10 min to produce a moderate skin injury. Oedema development peaked ca. 48 h following exposure, as determined by weighing ear biopsies. Histological observations at that time exhibited damage to the epidermis and dermis. An increase in prostaglandin E (PGE) was measured in skin homogenates, starting 8 h following exposure and lasting at least up to 48 h post-exposure. A topical treatment using the above anti-inflammatory mixture significantly reduced inflammatory parameters when applied up to 4 h following exposure. These parameters included extent of oedema, levels of PGE, area of clinical damage and extent of cytotoxic injury (vesications and damaged epithelial cells). Thus, a combination of a steroid and NSAID was found to be effective in reducing the intensity of HD skin injury and possibly shortening the time to full recovery. The treatment, however, did not prevent completely the ensuing cytotoxic processes in the epithelial layer.