摘要
目的：应用毒理学方法研究艾烟的一般毒性及特殊毒性,进行艾烟的安全性评价。
方法：实验选用全自动动态染毒装置进行体内动物实验：急性毒性实验和慢性毒性实验,该装置能实时监测和控制染毒腔内的温度、湿度、压差、氧气浓度以及受试烟雾的浓度,染毒环境稳定；以便携式PM2.5/PM10采样器PM10采样头采样艾烟中的可吸入颗粒物,二甲基亚砜溶解制备艾烟染毒液(Moxibustion Smoke Condensation, MSC),进行体外细胞及细菌实验：细胞毒性实验、染色体畸变实验、微核实验、细菌回复突变实验。
1急性毒性实验：以KM小鼠和Wistar大鼠为研究对象。KM小鼠为固定艾烟的浓度(60%和85%),以小鼠不同的染毒时间为剂量梯度,记录烟雾致小鼠死亡的染毒时间t,求得LT50(lethal concentration of50%, IC50); Wistar大鼠为固定2h的染毒时间,以不同的艾烟浓度(95%、90%、85%、80%、75%)为剂量梯度,记录不同剂量组大鼠的死亡只数,求得LT50(lethal time of50%, IT50)。分别可得出LD50(lethal dose of50%,ID50)。
2慢性毒性实验：对Wistar大鼠进行重复染毒,观察长期连续重复染毒艾烟所产生的毒性反应。Wistar大鼠随机分为四组,对照组(C组)、艾烟低剂量组(L组)、艾烟中剂量组(M组)、艾烟高剂量组(H组),观察艾烟染毒对动物的一般情况、体重、血液生化、血常规、尿常规、病理以及肺部相关的氧化、炎症、肺功能等指标的影响。
3细胞毒性实验：选用MTT的方法观察不同浓度的MSC抑制CHO细胞生长或对细胞产生不良反应的情况,观察艾烟引起细胞溶解和细胞死亡的实验,最终计算MSC对CHO细胞的半数抑制率IC50,为后期的体外实验提供数据。
4染色体畸变实验：以不同浓度的MSC染毒CHO细胞株,观察有丝分裂期的染色体畸变情况,考虑体内、外环境的影响,设S9活化(+S9)及非活化(-S9)的对照,组别设计为：对照组(KB)、KB+S9组、环磷酰胺+S9组(CP+S9)、丝裂霉素C组(MMC)、艾烟1/2LC50+S9组、艾烟1/4LC50+S9组、艾烟1/8LC50+S9组、艾烟1/2LC50-S9组、艾烟1/4LC50-S9组、艾烟1/8LC50-S9组,对各组染色体畸变的类型及细胞数进行统计。
5微核实验：以不同浓度的MSC染毒CHO细胞株,观察微核的形成情况,计算微核形成率,组别设计为：空白对照组(KB)、阳性对照组(CP)、溶剂对照组(DMSO)、1/2IC50、1/4IC50、1/8IC50,重复3次,观察微核形成率及是否具有剂量效应关系。
6鼠伤寒沙门氏菌突变回复实验：以平板掺入法观察MSC致鼠伤寒沙门氏菌株TA97、TA98、TA100和TA102基因突变情况,分别设62.5,125,250,500,1000μg/皿6个梯度,每组均做代谢活化(+S9)和非活化(-S9)的对照。不同菌株选用不同的代谢活化和非活化的阳性对照物,重复三次。观察结果在背景生长良好条件下,受试物组回变菌落数增加一倍以上(即回变菌落数等于或大于2乘以未处理对照数),并有剂量反应关系或至少某一测试点有可重复的并有统计学意义的阳性反应,即可认为该受试物诱变实验阳性。阳性结果至少应做三次测试,阴性结果至少进行二次测试,才能对受试物作出判定。
结果：艾烟相关的毒理学实验结果如下：
1急性毒性实验：艾烟急性毒性损伤肺脏及呼吸系统;KM小鼠LD50:45.41min,85%;209.96min,65%; Wistar大鼠LD50:86.274%,120min。
2慢性毒性实验：通过观察染毒期末大鼠的一般状况、血、尿常规及血生化,未发现因吸入艾烟引起的特异性的毒性反应,M、H组大鼠的体重降低、SOD消耗性降低、MDA增多、Th1/Th2细胞的效应因子均出现不同程度的下降,L组未引起毒性改变,恢复期M、H组也出现了大鼠TG、Glu增高、TP降低、Alb降低,综上我们认为L组未引起毒性改变,为NOAEL (no observed adverse effect level, NOAEL),以此数据建立相关的安全性评价。
3细胞毒性实验：MTT法观察艾烟的细胞毒性实验半数抑制浓度(lethal concentration of50%, IC50)为0.087mg/ml.
4染色体畸变实验：艾烟引起的染色体结构畸变包括染色单体型畸变和染色体型畸变二类。单体型畸变有：单体裂隙(ctg)、双间隙(csg)、单体断裂(ctb)、单体互换(cte)和单体缺失(ctd)；染色体型畸变有：双着丝粒体(dic)、环(ring)和无着丝粒断片(ace)。单体类型畸变中以单体裂隙、单体断裂和单体互换为多见,染色体型畸变以无着丝粒短片多见。染色体畸变细胞数和总畸变率一致,由高到低依次为CP+S9、艾烟+S9、MMC、 KB+S9、艾烟-S9组、空白对照组,艾烟使染色体畸变率增加,为潜在的染色体的断裂剂,具有潜在的诱变作用。
5微核实验：1/2IC50,1/4IC50,1/8IC50的MSC所诱发的微核形成率较阴性对照组增加,并且具有剂量效应关系,实验重复3次,可重复并且有统计学意义。
6鼠伤寒沙门氏菌突变实验：MSC在代谢活化后：TA98诱变阳性,在背景生长良好的条件下,TA98菌的回变菌落数在代谢活化后增加一倍以上,并有剂量反应关系,并有统计学意义,TA97、TA100、TA102诱变阴性；非代谢活化后均为阴性,此结果可以重复。因此,可以推论艾烟具有潜在的致基因突变性。
结论：艾烟毒理学研究结论如下：
1艾烟不属于危险废物,高浓度的艾烟持续染毒,使大小鼠死亡的主要原因为艾烟中大量的CO。
2艾烟的毒性与浓度有关,低浓度对大鼠的一般状况、代谢、免疫、血象、肝肾功能均无明显影响,中、高浓度反复染毒损伤肺及呼吸系统,对大鼠的代谢,免疫有一定的毒性,对血象、肝肾功能未见明显影响。实验得NOAEL为10%,艾烟的可吸入颗粒物的质量浓度为27.5mg/m3。根据动物实验艾烟的安全浓度外推到人,预期限制浓度为2.75mg/m3。
3随着艾烟浓度的增高及染毒时间的延长,艾烟可能具有遗传毒性,在使用艾灸进行治疗时应努力控制艾烟浓度及接触时间。
4目前的诊疗环境应注意通风,改善设施,提高艾灸治疗环境的安全性。
Objective:To study the acute, chronic toxicity and genetoxicity of moxa smoke and to evaluate its safety by principles of toxicology.
Methods:Animals were exposed in an automatic dynamic exposure devic, which display and control the exposed temperature, humidity, pressure, oxygen and the concentration of moxa smoke for acute toxicity and chronic toxicity study. Moxibustion Smoke was sampled by BHF7-MINIVOLTM-PM2.5and melted in DMSO and then used in vitro test, such as cytotoxicity test, chromosomal aberration tests, micronucleus test&Ames test.
1Acute toxicology study:KM mice were tested in exposure devic at60%(opacity) and80%for lethal time of50%(LT50); Wistar rats were randomly divided into five groups, were tested in exposure devic at moxa smoke dosages of75%,80%,85%,90%for2h, for lethal dose of50%(LD50).
2Chronic toxicity study:In Chronic toxicity study, rats were randomly divided into four groups to evaluate the toxic effects at different dosages in12weeks,24weeks after exposure and12weeks after post-exposure. Toxic reaction, dead rats number, tissue pathology, organ/body weight, haematological parameters, clinical chemistry parameters, routine urinalysis, and pulmonary function, expression of SOD, MDA, IL-2, IL-4, IL-10, IFN-y in lung was observed.
3Cytotoxicity test:Different consentration treated the Chinese hamster ovary cells (CHO) cells, which were cultured in vitro, and harvested24h after MSC treatment by using MTT method, and then obtained the IC50value in CHO, to provide data for the latter the experiment.
4Chromosomal aberration tests:To test the activity of MSC in vitro genetic toxicology test systems with the incidence of chromosomal aberrations in CHO. We incubated CHO with S9mix (+S9) and without S9mix (-S9) and harvested24h after treatment, and then prepared and analysed metaphase chromosome samples with addition of colchicine. Treatment group:control group-S9, mitomycin C-S9,1/2IC50MSC-S9,1/4IC50MSC-S9,1/8IC50MSC-S9, control group+S9, cyclophosphamide+S9,1/2IC50MSC+S9,1/4IC50MSC+S9,1/8IC50MSC+S9.
5Micronucleus test:To test the activity of MSC in vitro genetic toxicology test systems with the high frequency of micronucleus in CHO. We incubated CHO and harvested24h after treatment, and prepared and analysed samples with addition of cytochalasin B. Treatment group:control group, cyclophosphamide group,1/2IC50MSC,1/4IC50MSC.1/8IC50MSC.
6Ames test:To screen the activity of MSC in vitro genetic toxicology test systems in mutagenicity using the Ames'test. Using the plate incorporation assay, MSC were assayed for mutagenicity employing several histidine dependent salmonella strains (TA97,TA98, TA100and TA102) both with and without metabolic activation using a liver fraction (S9).Treatment group:control group-S9,62.5-S9,125-S9,250-S9,500-S9,1000-S9μg/dish, Positive control, control group+S9,62.5+S9,125+S9,250+S9,500+S9,1000+S9μg/dish, Positive control+S9. Each set up three plates. When a mutagen is added to the plate, the number of revertant colonies per plate increased, and usually is equal to or more than2times the number of spontaneously induced revertant colonies per plate, usually in a dose-related manner.
Results:Results of research on toxicology of moxa smoke were as follows:
1Acute toxicity study:In the acute study, the lethal dosage of50%(LD50) for a2h-exposure to moxa smoke was86.247%in wistar rats, and the lethal time of50%for85%and65%is45.41min and209.96min in KM mice.
2Chronic toxicity study:There were no deaths in the chronic study. There were, however, histopathological changes in the lungs in all exposed groups although there were no functional pulmonary changes compared to control. These histopathological alterations were unchanged at36weeks. No significant abnormalities in body weight at the low dosage were observed when compared to control. however, the middle and high dosage produced decreases in body mass but returned to normal at36weeks. No significant abnormalities in blood count, blood chemistry, or routine urinalysis were observed.
3Cytotoxicity test:For MSC the IC50value in Chinese hamster ovary cells (CHO) harvested24h after treatment obtained using MTS/PMS was0.087mg/ml.
4Chromosomal aberration tests:Chromosomal Aberration Tests:The incidence of chromosomal aberrations in CHO, in descending order, were CP+S9, MSC+S9, MMC+S9, control group+S9, MSC-S9, control group-S9. MSC has biological activity and showed some cytotoxicity at high-dose. MSC were positive when they were activated with the S9mix.
5Micronucleus test:1/2IC50MSC,1/4IC50MSC,1/8IC50MSC showed higher frequency of micronucleus in CHO compared to control group.
6Ames test:Without S-9, MSC showed mutagenic activity that was not enhanced, while, with S-9MSC were found to be mutagenic for strain TA98rather than TA97,TA100,TA102. Conclusion This Ames test screened MSC a mutagenic potential.
Conclusion:
1Moxa smoke is not hazardous waste, and continued exposure of high concentrations of moxa smoke killed rats and mice, mainly due to CO.
2the security of moxa smoke is up to the concentrations of it. Low concentration had no significant effect, while, medium and high concentrations damaged the lungs and respiratory system, affected metabolism, immune system and showed no significant effect on the blood cells, liver and kidney function. The low concentration is the NOAEL which is10%and with the respirable particulate matter level28.4mg/m3. For human the expected limit concentration is2.8mg/m3.
3with high concentration and long exposure time, the moxa smoke may have a genetic toxicity, the concentration and time of exposure should be controled.
4The current moxibustion environment should be improved in ventilation to enhance the security of it.
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