Differences in Airway Remodeling and Emphysematous Lesions between Rats Exposed to Smoke from New-Type and Conventional Tobacco Varieties.

Genes from Perilla frutescens and Ocimum basilicum were introduced into N. tabacum L. var. HHY via distant hybridization, and the new-type tobacco varieties "Zisu" and "Luole" were developed, with noticeable differences in chemical composition. Smoking is the leading cause of chronic obstructive pulmonary disease (COPD), and its pathogenesis is complex. In the present study, 48 male Sprague-Dawley (SD) rats were randomly divided into four groups, namely, the control, "HHY", "Zisu" and "Luole", and then exposed to fresh air/cigarette smoke (CS) for 30 days and 60 days. The COPD model was constructed, and their health hazards were compared and evaluated. CS from different tobacco varieties influenced rats in varying degrees at the tissue, cell and molecular levels. The rats in the "HHY" group showed obvious symptoms, such as cough and dyspnea, which were less severe in the "Zisu" and "Luole" groups. Pathological and morphological analyses, including scores, MLI, MAN, WAt/Pbm and WAm/Pbm, showed that "Zisu" and "Luole" caused less damage to the airways and lung parenchyma than "HHY". Significant increases in the numbers of total leukocytes and neutrophils in the BALF were found in "HHY" compared to those in "Zisu" and "Luole". Moreover, they caused less oxidative stress and apoptosis in lung tissues, as reflected by indicators such as ROS, MDA, T-AOC, GSH, the apoptotic index and the ratio of Bcl-2 to Bax. "Zisu" and "Luole" even altered the ratios of MMP-9/TIMP-1 and IFN-γ/IL-4 in lung tissues to a lesser degree. These differences between CS-exposed rats may be closely related to the altered expression of Nrf2, p38 MAPK and p-p38 MAPK. Changes in chemical composition via introducing genes from some medicinal plants may be an attractive strategy for tobacco harm reduction.

Tobacco introduced Perilla frutescens and Ocimum basilicum genes attenuates neutrophilic inflammation in lung tissues of COPD rats.

The new-type tobacco varieties "Zisu" and "Luole" were obtained by distant hybridization between N. tabacum L. var. HHY and Perilla frutescens and Ocimum basilicum, with obviously different chemical composition. Smoking is the major risk factor for COPD, characterized by neutrophil-dominant inflammation. In the present study, rat COPD model was established by cigarette exposure, and the health hazard of three varieties was compared by general condition observation, pathological and morphological evaluation, total and differential cell numeration, and characterization of major inflammatory mediators and MAPK/NF-κB pathway, etc. Rats in "HHY" group developed obvious symptoms such as cough, dyspnea, mental fatigue, etc., but these symptoms were obviously mitigated in "Zisu" and "Luole" groups. H&E staining analysis, including score, MLI, MAN, wt% and WA%, showed that "Zisu" and "Luole" significantly alleviated lung injury and the degree of airway remodeling and emphysema compared to "HHY". In BALF, the number of total leukocyte and the percent neutrophils in "Zisu" and "Luole" groups were evidently lower than "HHY" group. The levels of inflammatory mediators, such as IL-8, MPO, MIP-2, LTB4, TNF-α and neutrophil elastase, in "HHY" group were obviously higher than "Zisu" and "Luole" groups. The ROS-mediated NF-κB p65 and p38MAPK pathways may play an important role. Results indicated that tobacco introduced perilla and basil genes could remarkably attenuate recruitment, infiltration and activation of neutrophils and intervene in airway inflammation, retarding disease progression, especially "Zisu". Changes in chemical composition via breeding techniques may be a novel way for tobacco harm reduction.

Enhancing the potential for cadmium phytoremediation by introducing Perilla frutescens genes in tobacco.

To improve the potential of cadmium phytoremediation, distant hybridization between tobacco (Nicotiana tabacum L. var. 78-04), a high-biomass crop, and Perilla frutescens var. frutescens, a wild Cd-hyperaccumulator, was carried out, developing a new variety N. tabacum L. var. ZSY. Seedlings at the six-leaf stage were grown in hydroponics and treated with 0 (control), 10 µM, 180 µM, and 360 µM CdCl2 for 7 days; then, the differences in Cd tolerance and accumulation and physiological and metabolic responses were evaluated among "ZSY" and its parents. At high Cd dose, the growth of "ZSY," such as fresh weight, plant height, and root length, was evidently better than "78-04." In contrast to P. frutescens and "78-04," "ZSY" could accumulate more Cd in shoots than roots. Under the same treatment, "ZSY" accumulated greater amounts of Cd in both shoots (195-1523 mg kg-1) and roots (140-1281 mg kg-1) than "78-04" (shoots: 35-89 mg kg-1, roots: 39-252 mg kg-1), followed by P. frutescens (shoots: 156-454 mg kg-1, roots: 103-761 mg kg-1). BCF and TF values of "ZSY" reached 38-195 and 1.2-1.4, which were far higher than those of "78-04" (BCF: 2.2-35.3, TF: 0.35-0.9). Perilla frutescens was found with BCF and TF of 11-156 and 0.5-1.5. Cd stress obviously promoted the production of ROS and MDA in seedlings but reduced chlorophyll contents, especially in "78-04." As a response to Cd stress, "ZSY" had higher SOD and CAT activities when compared to P. frutescens and "78-04," while "78-04" produced more POD and proline than those of P. frutescens and "ZSY." Cd stress could affect the production and accumulation of alkaloids and phenolic compounds in root (endodermis and cortex) and mesophyll. At high Cd doses, P. frutescens and "ZSY" had more alkaloids in tissues than "78-04." Phenolic compounds in "78-04" were more obviously inhibited compared with P. frutescens and "ZSY." These secondary metabolites may play an important role in eliminating oxidative damage and enhancing Cd tolerance and accumulation in "ZSY" and P. frutescens. Results indicated that distant hybridization could be one of effective methods for introducing excellent genes from metal-hyperaccumulators into high biomass species, creating plants with superior phytoremediation potential.

The response of phenoloxidase to cadmium-disturbed hepatopancreatic immune-related molecules in freshwater crayfish Procambarus clarkii.

There has been extensive research on local and systemic oxidative stress and immunosuppression in cadmium exposed crustaceans, but the underlying mechanisms remain to be elucidated. Because of multiple functions of epithelial cells, such as storing and detoxifying heavy metals, producing and secreting immune-related molecules (i.e. hemocyanin, NF-κB and CBS/H2S et al.), hepatopancreas may play an important role in immune system. In the present study, as an indication of systemic and local immune status in crayfish Procambarus clarkii, the relationship between PO activities in haemolymph and levels of CBS/H2S/NF-κBp65 in hepatopancreas was evaluated following a 96 h exposure to sub-lethal Cd2+ concentrations (1/40, 1/8 and 1/4 of the 96 h LC50). The results indicated that there was a significant increase in ROS contents accompanied by markedly decreased THC and PO levels (P < 0.01) in a dose- and time- dependent manner. The evolutionarily conserved CBS and NF-κB p65 showed obvious difference (P < 0.01) (including cellular distribution and expression level) between the healthy and pathological conditions based on IHC analysis. Even small change of endogenous H2S content may be closely related to NF-κB p65 level and PO activity (P < 0.01). There was significantly negative correlation (P < 0.05) between PO activity and expression levels of CBS and NF-κB p65. Obviously, crayfish innate immunity was a highly complex network of various cells, molecules, and signaling pathways which operate, at least partly, through small signaling molecules such as H2S. ROS-mediated CBS/H2S/NF-κB pathway probably allowed hepatopancreas to inhibit PO activity under cadmium stress.

Copper-induced oxidative damage to the prophenoloxidase-activating system in the freshwater crayfish Procambarus clarkii.

Previous studies have demonstrated copper-induced proteins damage in gill and hepatopancreas of the freshwater crayfish Procambarus clarkii, but little information is available about its effects on key component of the innate defense in haemolymph. In the present study, we evaluated the relationship between oxidative carbonylation and prophenoloxidase-activating system (proPO-AS) activity, by exposing P. clarkii to sub-lethal concentrations (1/50, 1/12, 1/6 and 1/3 of the 96 h LC50) Cu(2+) up to 96 h. Six biomarkers of oxidative stress, i.e. reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), protein carbonyl (PC), malondialdehyde (MDA) and DNA-protein crosslinks (DPCs), and six indicators of immune status, i.e. total hemocyte counts (THCs), differential hemocyte counts (DHCs), hemocyanin (HC), prophenoloxidase (proPO), serine protease (SP) and phenoloxidase (PO), were determined in haemolymph. The results indicated that there was a significant increase (P < 0.05) in the levels of ROS, PC, MDA and DPCs accompanied by markedly decreased (P < 0.05) activities of proPO, SP, PO and HC in a dose and time dependent manner. The significant and positive correlations (P < 0.01) between ROS production and the formation of PC, MDA and DPCs were observed in crayfish at 96 h. There was a significant negative correlation (P < 0.01) between the levels of protein carbonyls and the activities of proPO and SP in hemocyte lysate supernatant and PO and HC in haemolymph. Carbonylated proteins may be recognized not merely as a specific signal in oxidative stress pathways but also as a "non-self" molecule in proPO-AS. In crayfish species, copper-catalyzed protein carbonylation may be one of the main mechanisms for immunity dysfunction in proPO-AS.

Oxidative damage of hepatopancreas induced by pollution depresses humoral immunity response in the freshwater crayfish Procambarus clarkii.

Previous studies provide evidences for the possible oxidative damage of toxic environmental pollutants to tissue protein in fish and amphibian, but little information is available about their effects on immunity response in crustacean. In the present study, we evaluated the relationship between oxidative damage and immune response induced by both typical pollutants (viz. copper and beta-cypermethrin), by exposing the freshwater Procambarus clarkii to sub-lethal concentrations (1/40, 1/20, 1/10 and 1/5 of the 96 h LC50) up to 96 h. Five biomarkers of oxidative stress, i.e. reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and protein carbonyl in hepatopancreas, and two immune factors, i.e. phenoloxidase (PO) and hemocyanin in haemolymph were determined. The results indicated that there was a significant increase (P < 0.05) in the contents of ROS, MDA and protein carbonyl accompanied by markedly decreased (P < 0.05) PO and hemocyanin levels in a dose and time dependent manner. The significant and positive correlation (P < 0.01) between protein carbonyls induction and MDA formation was observed in crayfish hepatopancreas at 96 h. The production of these protein carbonyls could significantly depress (P < 0.01) the levels of phenoloxidase and hemocyanin in hemolymph. Higher contents of ROS enhanced the risk of lipid peroxidation, protein carbonylation and immunosuppression of crayfish, and hepatopancreas might play an important role in immune system of crustaceans. Protein oxidation may be one of the main mechanisms for pollution-induced immunotoxicity in P. clarkii.

Enhancement of cadmium tolerance and accumulation by introducing Perilla frutescens (L.) Britt var. frutescens genes in Nicotiana tabacum L. plants.

The tobacco has the genetic potential to remove toxic metals from the soil. To develop hyperaccumulating tobacco plants, distant hybridization between tobacco (Nicotiana tabacum L.), a high-biomass crop, and Perilla frutescens (L.) Britt var. frutescens, a newfound Cd-hyperaccumulator species, was carried out using a novel method viz. pollination following grafting. Their hybrid nature was preliminarily confirmed by phenotype, isozyme pattern, random amplified polymorphic DNA (RAPD) and metabolites analysis. About 120 putative F2 hybrids derived from the cross-combination [(N. sylvestris Speg. & Comes rootstock + N. tabacum L. var. 78-04 scion) × P. frutescens (L.) Britt var. frutescens] were then subjected to up to 300 µM CdCl2 in hydroponic conditions for 10 days. Results showed five seedlings were more resistant to Cd than female parent and accumulated 314.6 ± 99.9 mg kg(-1) Cd in their aerial biomass, which was 5.7 times greater than that in "78-04" tobacco (47.2 ± 3.56 mg kg(-1)) (P ≤ 0.05). Two of these seedlings exceeded male parent P. frutescens in the Cd concentration of shoots and reached 424 and 396 mg kg(-1), which was 13% and 6% greater for that of perilla (374.2 ± 10.38 mg kg(-1)), respectively. Compared with parents, two other F2 hybrids tended to accumulate more Cd in the root with bioconcentration factor (BCF) 7.05 and 5.17, respectively. Only one hybrid showed lower Cd concentration but transferred Cd more effectively from the root to the shoot than parents and other F2 hybrids, with the maximum translocation factor (TF) value 1.37. These indicated that the introduction of P. frutescens genes could obviously enhance the cadmium tolerance and accumulation of superior individuals.

Oxidative damage induced by copper and beta-cypermethrin in gill of the freshwater crayfish Procambarus clarkii.

Copper (Cu) and pyrethroid are common contaminants found in the aquatic environment, and their potential toxicological effects on aquatic organisms have received extensive attention. However, the impact on crayfish species of exposure to the two chemicals are still largely unknown. The current study assessed the sublethal toxicities induced by Cu and commercial formulation of beta-cypermethrin in the freshwater crayfish Procambarus clarkii. The static test method of acute toxicity test was used. Five biomarkers of oxidative effects, i.e. reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and protein carbonyls, and pathologic changes were determined. The results demonstrated that there was a significant increase in the contents of ROS, MDA and protein carbonyls accompanied by markedly decreased SOD and CAT activities in a concentration-dependent manner. The linear relationship between protein carbonyls induction and MDA formation was observed evidently in crayfish gills at 96h. Higher contents of ROS enhanced the risk of lipid peroxidation and protein carbonylation, thus impacting vital physiological functions of gills. The results indicated that protein oxidation may be one of the main mechanisms of pollution-induced toxicity to crayfish gills. Protein carbonyl levels could be considered as a potential biomarker of exposure to environmental contaminants.

Histological alterations and immune response in the crayfish Procambarus clarkii given rVP28-incorporated diets.

There is growing evidence that recombinant VP28 protein (rVP28) can significantly enhance immune response and disease resistance against white spot syndrome virus (WSSV) in shrimp, although the underlying mechanisms have not been entirely clarified yet. The aim of this study was to determine the effect of rVP28 on histological alterations and WSSV-induced apoptosis in crayfish Procambarus clarkii. Crayfish were fed commercial diets supplemented with different doses of HyNPV-VP28 infected pupae (rVP28-hp) for 4 weeks. Results showed that rVP28-hp may be used as a safe and effective source of medicinal proteins in aquaculture when supplemented in diet at low dose (10 g kg(-1) and 50 g kg(-1)), which could obviously reduce the percentage of apoptotic cells in stomach, gut and hepatopancreas tissues induced by the WSSV challenge and showed the relative percent survival (RPS) of 82.2% and 94.4%, respectively. But rVP28-hp would be detrimental to crayfish survival and decrease resistance to WSSV infection at the high dose (100 g kg(-1) and 200 g kg(-1)), with the cumulative mortality of up to 48.2% and 56.6% after WSSV challenge, respectively. During a 28-d feeding period, the survival rate of crayfish was only 54.5%-75.6%, and histopathological observation showed that one of the principal lesions was serious cell swelling, vacuolar degeneration and necrosis in hepatopancreatic epithelia and myocardial cells. These results suggested that rVP28-hp can influence the immune functions of crayfish in a dose-dependent manner, and the rVP28-hp at the dose of 50 g kg(-1) was recommended to prevent WSSV in crayfish culture.