Guojun Zhou

Comparison between two kinds of cigarette smoke condensates (CSCs) of the cytogenotoxicity and protein expression in a human B-cell lymphoblastoid cell line using CCK-8 assay, comet assay and protein microarray

The differences of the cytogenotoxicity and proteins expression of human B-cell lymphoblastoid cells exposed to cigarette smoke condensates (CSCs) from two kinds of cigarettes were detected with CCK-8 assay, comet assay, protein microarray and western blot assay in vitro. Human B-cell lymphoblastoid cell line was exposed to CSCs from two cigarettes (which delivers approximately 3mg tar, 0.3mg nicotine, 3mg CO per cigarette for cigarette 1 and 15mg tar, 1.3mg nicotine, 15mg CO per cigarette for cigarette 2), and the exposure doses were 2.5, 5.0, 7.5, 10.0 and 12.5x10(-3)cigarettes/ml of CSCs for 24h in CCK-8 assay, 6.0, 8.0, 10.0, 12.0 and 14.0x10(-3)cigarettes/ml of CSCs for 4h in comet assay, and 10.0x10(-3)cigarettes/ml of CSCs for 4h in protein levels analysis. The results of CCK-8 assay and comet assay in the present study suggested that the cytogenotoxicity in cigarette 2 group was significantly higher than that in cigarette 1 group. The results of protein microarray and western blot assay showed that there were the differences of the expression levels of four proteins (i.e., RAR-beta, 14-3-3 sigma, XPF, and p57(Kip2) Ab-7) between cigarette 1 group and cigarette 2 group. Hence, it is possible that the RAR-beta, 14-3-3 sigma, XPF, and p57(Kip2) Ab-7 proteins serve as the molecular biomarkers in studying the cytogenotoxicity induced by CSCs.

Studying the cyto-genotoxic effects of 12 cigarette smoke condensates on human lymphoblastoid cell line in vitro.

In the present study, the cyto-genotoxic effects of 12 CSCs prepared from a diverse set of cigarettes on human B lymphoblastoid cells were compared using five in vitro assays. The cells were exposed to CSCs at doses of 2.5, 5.0, 7.5, 10.0, and 12.5 x 10(-3)cigarette/ml for 24h in neutral red uptake and CCK-8 assays, at doses of 1.0, 2.0, 3.0, 4.0, and 5.0 x 10(-3)cigarette/ml for 3h in cell apoptosis assay, at doses of 6.0, 8.0, 10.0, 12.0, and 14.0 x 10(-3)cigarette/ml for 4h in comet assay, and at doses of 1.0, 2.0, 4.0, 6.0, and 8.0 x 10(-3)cigarette/ml for 4h in micronucleus assay. The potency of 12 CSCs to induce corresponding toxic effects in each assay was calculated, and the correlations between the results in five assays were analyzed. Our investigation showed that the results of 12 CSCs in CCK-8 and cell apoptosis assays were positive, the results of 11 CSCs in neutral red uptake and comet assays were positive, and 9 CSCs could induce significantly the micronuclei in micronucleus assay. It was found that the potency to induce the cytotoxic effects among 12 CSCs ranged 9.694 folds in neutral red uptake assay and 6.43 folds in CCK-8 assay, the potency to induce cell apoptosis among 12 CSCs ranged 8.191 folds, the potency to induce DNA damage among 12 CSCs ranged 29.199 folds, the potency to induce micronuclei among 12 CSCs ranged 5.879 folds. Moreover, the good correlations were found between any two assays. It was suggested that the cyto-genotoxicity of CSCs from different brands of cigarettes varied greatly, comet assay might be a sensitive assay in assessing the genotoxicity induced by CSCs.

Genetic variation in alkaloid accumulation in leaves of Nicotiana.

Alkaloids are plant secondary metabolites that are widely distributed in Nicotiana species and contribute greatly to the quality of tobacco leaves. Some alkaloids, such as nornicotine and myosmine, have adverse effects on human health. To reduce the content of harmful alkaloids in tobacco leaves through conventional breeding, a genetic study of the alkaloid variation among different genotypes is required. In this study, alkaloid profiles in leaves of five Nicotiana tabacum cultivars and Nicotiana tomentosiformis were investigated. Six alkaloids were identified from all six genotypes via gas chromatograph-mass spectrometry (GC-MS). Significant differences in alkaloid content were observed both among different leaf positions and among cultivars. The contents of nornicotine and myosmine were positively and significantly correlated (R2=0.881), and were also separated from those of other alkaloids by clustering. Thus, the genotype plays a major role in alkaloid accumulation, indicating a high potential for manipulation of alkaloid content through traditional breeding.

Rapid determination of the volatile components in tobacco by ultrasound‐microwave synergistic extraction coupled to headspace solid‐phase microextraction with gas chromatography‐mass spectrometry

An ultrasound-microwave synergistic extraction coupled to headspace solid-phase microextraction was first employed to determine the volatile components in tobacco samples. The method combined the advantages of ultrasound, microwave, and headspace solid-phase microextraction. The extraction, separation, and enrichment were performed in a single step, which could greatly simplify the operation and reduce the whole pretreatment time. In the developed method, several experimental parameters, such as fiber type, ultrasound power, and irradiation time, were optimized to improve sampling efficiency. Under the optimal conditions, there were 37, 36, 34, and 36 components identified in tobacco from Guizhou, Hunan, Yunnan, and Zimbabwe, respectively, including esters, heterocycles, alkanes, ketones, terpenoids, acids, phenols, and alcohols. The compound types were roughly the same while the contents were varied from different origins due to the disparity of their growing conditions, such as soil, water, and climate. In addition, the ultrasound-microwave synergistic extraction coupled to headspace solid-phase microextraction method was compared with the microwave-assisted extraction coupled to headspace solid-phase microextraction and headspace solid-phase microextraction methods. More types of volatile components were obtained by using the ultrasound-microwave synergistic extraction coupled to headspace solid-phase microextraction method, moreover, the contents were high. The results indicated that the ultrasound-microwave synergistic extraction coupled to headspace solid-phase microextraction technique was a simple, time-saving and highly efficient approach, which was especially suitable for analysis of the volatile components in tobacco.

Chemical constituents of tobacco smoke induce the production of interleukin-8 in human bronchial epithelium, 16HBE cells

BackgroundInterleukin-8 (IL-8) functions as a major chemoattractant and plays pivotal roles in the initiation and development of chronic obstructive pulmonary disease (COPD), and tobacco smoke is a most risk factor contributing to the development of COPD. Hence, we have screened some of the tobacco smoke-derived chemical compounds that potentially induce the production of IL-8 in human bronchial epithelium, 16HBE cells.MethodsTwenty-eight hazardous smoke components belonging to 9 classes including nicotine, ammonia, aromatic amines, polycyclic aromatic hydrocarbons, phenols, carbonyls, hydrocyanic acid, nitrosamines and other volatile organics were used in the experiments. Proliferation of 16HBE cells was determined by cell counting kit-8 kit, luciferase activity was measured in IL-8 reporter gene-expressing 16HBE cells, and IL-8 levels in culture supernatants were quantified by enzyme-linked immunosorbent assay.ResultsAt the non-toxic dosages, chemical compounds belonging to nicotine, aromatic amines, benzopyrene, phenols, aldehydes, and some other volatile organics dose-dependently increased IL-8 reporter gene expression. Consistently, the representative compounds belonging to nicotine, aromatic amines, benzopyrene, phenols, aldehydes, and some other volatile organics significantly and dose-dependently increased IL-8 levels in the culture supernatants of 16HBE cells, among these compounds, benzopyrene is a most potent stimulator for inducing IL-8 production.ConclusionsThe present study has identified particular tobacco smoke constituents responsible for inducing the IL-8 production in human bronchial epithelium, which might help shed light on the pathogenesis of tobacco smoke-induced COPD.

Ginkgolide B functions as a determinant constituent of Ginkgolides in alleviating lipopolysaccharide-induced lung injury

Ginkgolides are the major bioactive components of Ginkgo biloba extracts, however, the exact constituents of Ginkgolides contributing to their pharmacological effects remain unknown. Herein, we have determined the anti-inflammatory effects of Ginkgolide B (GB) and Ginkgolides mixture (GM) at equivalent dosages against lipopolysaccharide (LPS)-induced inflammation. RAW 264.7 cell culture model and mouse model of LPS-induced lung injury were used to evaluate in vitro and in vivo effects of GB and GM, respectively. In RAW 264.7 cells, GB and GM at equivalent dosages exhibit an identical capacity to attenuate LPS-induced inducible nitric oxide synthase mRNA and protein expression and subsequent NO production. Likewise, GB and GM possess almost the same potency in attenuating LPS-induced expression and activation of nuclear factor kappa B (p65) and subsequent increases in tumor necrosis factor-α mRNA levels. In LPS-induced pulmonary injury, GB and GM at the equivalent dosages have equal efficiency in attenuating the accumulation of inflammatory cells, including neutrophils, lymphocytes, and macrophages, and in improving the histological damage of lungs. Moreover, GB and GM at equivalent dosages decrease the exudation of plasma protein to the same degree, whereas GM is superior to GB in alleviating myeloperoxidase activities. Finally, though GB and GM at equivalent dosages appear to reduce LPS-induced IL-1β mRNA and protein levels and IL-10 protein levels to the same degree, GM is more potent than GB to attenuate the IL-10 mRNA levels. Taken together, this study demonstrates that GB functions as the determinant constituent of Ginkgolides in alleviating LPS-induced lung injury.

Characteristic fragmentation behavior of tobacco‐specific N‐nitrosamines using electrospray ionization multistage tandem mass spectrometry incorporating deuterium labeling

RATIONALE Tobacco-specific N-nitrosamines (TSNAs) mainly include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT) that are formed from tobacco alkaloids during the curing process and contained in tobacco and tobacco smoke. They are linked with carcinogenesis. Analytical methods for quality control of products and determination of their metabolites are therefore of great importance. METHODS The characteristic fragmentation behaviors of tobacco-specific TSNAs have been studied by electrospray ionization multistage tandem mass spectrometry. The deutero-labeled TSNA compounds have also been employed to clarify the fragmentation mechanism, which further confirms the proposed fragmentation patterns. RESULTS Detailed analysis of the resultant fragments shows there are two different kinds of fragmentation patterns with the general fragment backbone of pyrrolidine or piperidine rings. In one route, pyrrolidine or piperidine rings undergo direct fragmentation and form some stable intermediates without affecting the parent rings. The other, however, involves ring opening and then ring closure at the pyridine-2 carbon atom to form multi-membered rings. CONCLUSIONS This characteristic fragmentation behavior therefore provides useful information on identification of TSNAs that may be used to monitor such kinds of compound in the biological metabolism.