Histogenesis Of Lung Adenocarcinoma Induced By Aflatoxin G₁ In Mice And The Effects Of AFG₁ On Alveolar TypeⅡ Cells

Aflatoxin G₁ (AFG₁) is one of the members of aflatoxins, the toxic metabolites produced by Aspergillus flavus, etc. Studies showed that AFG₁ was the most frequently detected contaminating mycotoxins in the foodstuffs of the high incidence areas of esophageal and lung cancers in Taihang mountain area in north China.Acting as progenitor cells to reform the alveolar epithelium after lung injury, alveolar type II cells (AT-II) contribute to the innate cellular immune response against airbrone pathogens. Repeated injury, repair and proliferation of AT-II induced by environmental carcinogenic agents such as smoking, quartz, fungi, etc, may finally result in the development of carcinoma in lung. Both direct and indirect mechanisms involved in injuries of lung tissues by environmental toxicants and carcinogens exposed to airway epithelium. The structural and functional changes of AT-II may form the basis for the carcinogenesis of lung tissue. It was confirmed that many adenocarcinomas of lung induced by environmental toxicants and carcinogens exposed to airway epithelium arised from alveolar type II cells.Our previous animal experiment studies showed that AFG₁ was carcinogenic. Oral administration of AFG₁ could induce lung adenocarcinomas in several experiment animals including mouse, rat, etc. Up to now, the studies of the effects of AFG₁ on carcinogenesis of lung have been limited to cancer inducing experiments and the further confirmation of the causative role of AFG₁ exposure to the carcinogenesis of lung tissue. No work has been done on the histogenesis of lung adenocarcinoma induced by oral administration of AFG₁. Few works have been involved in the acute effects of AFG₁ on the lung tissues and AT-II in vivo and in vitro.To further explore the putative mechanisms of carcinogenesis of AFG₁, the following works were carried out in this study. The histogenesis of the lung adenocarcinomas induced by AFG₁ was studied by determination of the phenotype of the adenocarcinoma cells with immunohistochemical staining of AT-II specific marker, SP-C and the Clara cell specific marker, CC-10 at protein level. The effects of acute AFG₁ treatment on the lung tissues and AT-II were studied both in vivo and in vitro. The effects of JNK signaling pathways on the injury of AT-II cell line-A549 cells induced by AFG₁ were also studied.The study includes the following five parts:1 Histogenesis of lung adenocarcinoma induced by oral adminstration of AFG₁ in miceObjective: To study the histogenesis of lung adenocarcinoma induced by AFG₁ and explore the effects of oral administration of AFG₁ on SP-C and PCNA expression of alveolar epithelial cells of lung in NIH mice.Methods: Paraffin embedded tissue blocks of 9 adenocarcinoma of lung induced by oral administration of AFG₁ as well as 15 lung tissues of the mice treated intragastrically by gavage with 3μg·kg^-1 and 30μg·kg^-1 AFG₁ for 58 weeks and 12 normal mice lung tissues in control group treated with normal saline were included in this study. The phenotype of the lung adenocarcinomas was determined by immunohistochemical expression of SP-C and CC-10 at protein level. The expression of P53 and Ras P²¹ in 9 cases of the lung adenocarcinomas was studied with immunohistochemical staining. The expression of SP-C and PCNA in lung tissues of 24 mice in AFG₁ group and that in lung tissue of 12 mice in control group was determined by immunohistochemical staining method.Results:1.1 The expression of SP-C and CC-10 in the lung adenocarcinomas induced by AFG₁The positive expression of SP-C was found in all the lung adenocarcinomas, while no expression of CC-10 could be seen in the 9 cases. Thus, the results in this study reveals that the lung adenocarcinomas induced by AFG₁ in NIH mice arise from alveolar type II cells1.2 The expression of P53 and Ras P²¹ in the lung adenocarcinomas induced by AFG₁Immunohistochemical results showed that no positive expression of mutant P53 and Ras P²¹ at protein level could be found in the lung adenocarcinomas.1.3 Effect of AFG₁ on the expression of SP-C and PCNA in alveolar epithelial cells in miceThe immunohistochemical labelling index of SP-C of alveolar epithelial cells in AFG₁ 3μg·kg^-1 and AFG₁ 30μg·kg^-1 group were 31.63±5.51% and 33.58±4.84% respectively, which were significantly higher than that in control group (19.72±1.92%, p<0.01). The labelling index of PCNA of alveolar epithelial cells in AFG₁ 3μg·kg^-1 and AFG₁ 30μg·kg^-1 group were also significantly higher than that in control group (p<0.01). These results suggest oral administration of AFG₁ can significantly increase the expression of SP-C in AT-II and promote proliferation of alveolar epithelial cells in NIH mice.2 Effects of intratracheal administration of aflatoxin G₁ on lung tissues in rats.Objective: To explore the effects of single intratracheal administration of aflatoxin G₁ on rat lung tissuesMethods: One hundred and twenty male SD rats weighting 110-130g were randomly divided into three groups: AFG₁ group, solvent control group and control group, 40 rats in each group. The experimental rats in the three groups were intratracheally administrated respectively with AFG₁ (30μg·kg^-1body weight), DMSO and saline. The rats were sacrificed 1, 3, 7 and 14d after AFG₁ treatment respectively. Bronchoalveolar lavage fluid (BALF) was collected for LDH and AKP release assay using Detection Kit with biochemical method. The lung tissues specimens were fixed in 2.5% glutaraldehyde and washed in PBS (0.1mol·L^-1, pH7.2) overnight and the ultrastructural changes of lung tissues were studied with scanning electron microscopy (SEM). Representative tissues specimens were fixed in 4% phosphate-buffered paraformaldehyde, embedded in paraffin and sectioned. The expression of TNF-αat mRNA level and NF-κB protein of lung tissues was studied with in situ hybridization and immunohistochemical staining method respectively. The concentrations of reactive oxygen species (ROS) of lung tissues were determined with biochemical method.Results:2.1 Effects of AFG₁ on LDH and AKP activity in BALF of ratsNo difference in LDH and AKP activity of BALF between the rats in solvent control group (DMSO) and control group was found. LDH and AKP activity of BALF in rats 1, 3and 7 d after AFG₁ treatment was significantly increased as compared to that in corresponding DMSO group (p<0.01). The LDH and AKP activity restored to control level 14 d after AFG₁ treatment. These results suggest that AFG₁ may cause reversible injury in rat lung tissues.2.2 Effects of AFG₁ on The pathologial changes of lung tissuesNo pathological changes were found in the rat lung tissues in control and DMSO group for each treat time point. The thickening of alveolar septum and lymphocyte and macrophage infiltration could be found in lung tissues after AFG₁ treatment.Injury changes such as, alveolar septum thickening, alveolar wall swelling changes, disappearance of microvilli in AT-II could be seen in lung tissues of rats 3, 7 and 14d after AFG₁ treatment by microscopical observation using SEM. These results further suggest that intratracheal administration of AFG₁ may cause ultrastructural injury changes of rat lung tissues.2.3 Effects of AFG₁ on the expression of TNF-αmRNA in rats lung tissuesIn situ hybridization results showed that the positive expression percentages of TNF-αmRNA in the alveolar cells in lung tissues of rats 1, 3, 7 and 14d after AFG₁ treatment were 22.1±4.0%,20.3±4.2%,32.3±4.2% and 24.3±3.8% respectively, which were all significantly higher than those of their corresponding DMSO groups 1, 3, 7 and 14 d after DMSO treatment, (1.2±0.3%,1.1±0.2%,1.3±0.4% and 1.0±0.4%, p<0.01).2.4 Effects of AFG₁ on the expression of NF-κB protein in rats lung tissues Prominent staining for NF-κB in bronchi epithelial cells could be seen in control, DMSO and AFG₁ treated groups. No positive expression of NF-κB at protein level could be found in the alveolar cells after AFG₁ treatment. These results suggest that intratracheal administration of AFG₁ may have no effect on the expression of NF-κB in rat lung tissues.2.5 Effects of AFG₁ on concentration of reactive oxygen species in lung tissuesExcept the rats 1d after AFG₁ treatment, the concentrations of ROS of lung tissues in rats 3, 7 and 14d after AFG₁ treatment were (53.4±12.4),(42.7±10.8) and (47.2±11.0) mol·min^-1·gprot^-1 , which were all significantly lower than those in their corresponding DMSO groups 3, 7 and 14 d, {(61.7±4.2), (66.3±5.7) and(64.3±5.4)mol·min^-1·gprot^-1, p<0.05}. Intratracheal administration of AFG₁ may decrease the antioxidant ability in rat lung tissues.3 Effects of intratracheal administration of Aflatoxin G₁ on lung alveolar type II cells in ratsObjective: To explore the effects of single intratracheal administration of Aflatoxin G₁ on structure and function of rat lung alveolar typeâ…¡cells.Methods: The rats in each group as described in part 2 were sacrificed 1, 3, 7 and 14d after AFG₁ treatment and parts of lung tissues specimens were used for the studies. The lung tissues specimens were fixed in 4 % glutaraldehyde and washed in PBS (0.1mol·L^-1, pH7.2) overnight and the ultrastructural changes of AT-II in lung tissues were studied with transmission electron microscopy (TEM). Immunohistochemical stainning was uesd to detect the expression of SP-C in rat lung tissues. The lung tissues fixed in 70% ethanol were used for preparation of single cell suspension and flow cytometric analysis of the expression of SP-C. Fresh lung tissues (200mg) for Western Blot was first homogenized in 1ml lysis buffer and then the total protein was extracted from the lung tissues and stored at -80℃. The expression of SP-C was determined by Western blot. The expression of SP-C and SP-A at mRNA level was detected by semi-quantitative RT-PCR. Results:3.1 Effects of AFG₁ on the ultrastructural changes of AT-II in rats lung tissuesTransmission electron microscopic observation revealed some injury changes in AFG₁ treated alveolar typeâ…¡cells, e.g. turgid and evacuated lamellar bodies, vacuolar degeneration of mitochondria with loss of crista structure, disappearance of microvilli, etc.3.2 Effects of AFG₁ on the expression of SP-C in the alveolar cellsThe results showed that 1d, 3d and 14d after AFG₁ treatment, no significant differences were found in SP-C labeling index of the alveolar cells between the rats in AFG₁ group and that in the corresponding DMSO group. But 7d after AFG₁ treatment, the immunohistochemical labelling index of SP-C of the alveolar cells on the rats in AFG₁ treatment Group was significantly lower than that of the DMSO group (9.10±1.28%, vs 13.34±2.31%, p<0.05).FCM results showed that 1, 3d and 14d after AFG₁ treatment, there was no difference in the expression of SP-C protein between AFG₁ treated groups and the corresponding DMSO groups. It was noted that 7d after AFG₁ treatment, the FI value of SP-C protein expression was significantly decreased in AFG₁ treated group (p<0.05).Similar to the results with immunohistochemical and FCM methods, Western blot also confirmed a significant decrease in SP-C expression at protein level 7d after AFG₁ treatment.Thus, the results in this study suggested that AFG₁ could cause decrease of SP-C expression in a time specific way.3.3 Effects of AFG₁ on the expression of SP-C mRNA in rats lung tissuesThe expression of SP-C mRNA in lung tissues of the rats 1 and 3d after AFG₁ treatment was not significantly changed as compared with that of conresponding DMSO rats. However, the expression of SP-C mRNA in lung tissues of rats was significantly decreased 7 days after AFG₁ treatment as compared with that in corresponding DMSO 7d group (p<0.01). The expression of SP-C mRNA in lung tissues of rats restored to normal level 7 days after AFG₁ treatment.3.4 Effects of AFG₁ on the expression of SP-A mRNA in rat lung tissuesExcept the rats 1d after AFG₁ treatment, the ratios of SP-A mRNA expression in AFG₁ treated groups 3 and 7 d after AFG₁ treatment were 0.72±0.09 and 0.43±0.12 respectively, which was significantly lower than those in the solvent groups at the same time points (1.03±0.21 and 1.06±0.1, p<0.01). The expression of SP-A mRNA in lung tissues of rats could also restore to normal level 14 days after AFG₁ treatment.The results further revealed AFG₁ treatment could cause reversible injury in a time specific way.4 Effects of aflatoxin G₁ on the primarily cultured rat lung alveolar type-II cellsObjective: To further explore the putative effects of aflatoxin G₁ on the possible target cells, primarily cultured AT-II from SD rats.Methods:The primarily cultured cells were resuspended in 10¹5mL DMEM medium supplemented with 20% FCS. Then AT-II cells were purified as follows, the cells were transferred to 0.1g·L^-1 rat IgG-coated (diluted in Tris buffer solution) Petri dishes (10cm) and AT-II was purified by adherence of macrophages to the dishes for 3 h of incubation. After 3h incubation with rat IgG, the cells were removed and centrifuged and then were seeded at (1²)×109L^-1 in culture flasks (8 ml), 24-well plates and 96-well plates with DMEM medium supplemented with 20% FCS at 37℃and 5% CO2. The medium of AT-II was replaced by new DMEM medium supplemented with 10% FCS 24 later and cultured for 12 h.After culture for 12 h, the primarily cultured cells were randomly divided into 5 groups: control, solvent control, AFG₁ 0.5 mg·L^-1, AFG₁ 1.0 mg·L^-1 and AFG₁2.0 mg·L^-1. AFG₁ were added to the medium at final concentrations of 0.5, 1.0 and 2.0mg·L^-1 as AFG₁ treated groups, DMSO was added to the medium of solvent control group and saline was added to the medium of control group. The cells were incubated with AFG₁, DMSO, or saline for 24 h. Cell viability for AT-II cells in vitro was assessed with MTT assay. The activities of lactate dehydrogenase (LDH) and alkaline phosphotase (AKP) in supernatant of AT-II cells in 24-well plates were evaluated by biochemical method. The cultured cells were harvested and fixed in 2.5% glutaraldehyde and ultrathin sections were prepared and stained by uranyl acetate-leadcitrate, the ultrastructural changes of AT-II were observed under transmission electron microscopy (TEM). The concentrations of intracellular free [Ca²⁺] ([Ca²⁺]i) of AT-II in 24-well plates loaded with Fluo-3/AM were observed under confocal laser scanning microscopy (CLSM). The immunocytochemical expression of SP-C of AT-II was determined by CLSM. The cultured cells were centrifuged and fixed in 70% ethanol for preparation of single cell suspension and used for flow cytometric analysis of the expression of SP-C.Results:4.1 Identification of AT-II in vitroThe specificity of the isolated and cultured AT-II was evaluated using TEM and alkaline phosphatase (AKP) histochemical staining. The specific ultrastructural structure - lamellar bodies could be seen in the cultrued alveolar type-II cells. AKP was positive in the cultured cells. Thus, the results showed that the isolated cells in culture in this study were AT-II.4.2 Effects of AFG₁ on the injuries of AT-II in vitroMTT assay showed that survival rates of AFG₁ 0.5, 1.0 and 2.0 mg·L^-1 group was 88±3%,80±9% and 72±8% respectively, which was significantly lower than that in DMSO group (101±2%, p<0.01). The activities of LDH and AKP in supernatant were significantly increased after AFG₁ treatment. The results suggest that AFG₁ may cause proliferation inhibition and injuries in cultured rat lung alveolar type-II cells.4.3 Effects of AFG₁ on the ultrastructure of AT-II in vitroInjury changes at ultrastructural level, such as, turgid and evacuated lamellar bodies, vacuolar degeneration of mitochondria were observed in AFG₁-treated cells under TEM. Exposure of AFG₁ could cause direct injury at structural level in cultured rat AT-II cells.4.4 Effects of AFG₁ on the concentrations of [Ca²⁺]i of AT-II in vitroThe [Ca²⁺]i was measured by CLSM and a significant elevation of [Ca²⁺]i was observated in AFG₁-treated cells. The concentrations of [Ca²⁺]i in 0.5, 1.0 and 2.0 mg·L^-1 AFG₁ group were 200±21, 225±14 and 229±12 respectively, which were significantly higher than that in DMSO group (161±28, p<0.01). A significant dose- depended response correlation could be found between AFG₁concentrations and [Ca²⁺]i (r=0.849, p<0.01).4.5 Effects of AFG₁ on the expression of SP-C protein in AT-II in vitroGreen fluorescence of SP-C protein expression was visualized by CLSM in ecah immunocytochemical stained sample. Fluorescence intensity of SP-C expresson in all AFG₁-treated groups (0.5, 1.0 and 2.0 mg?L^-1)was significantly lower than that in DMSO group (225±18,209±14 and 195±13 vs 243±8, p<0.01).FCM analysis showed that FI of SP-C protein expression in 0.5, 1.0 and 2.0 mg?L^-1 AFG₁ group was 0.91±0.04, 0.88±0.06 and 0.76±0.05 respectively, which was significantly lower than that in DMSO group (0.99±0.06, p<0.01). The results indicated that exposure of AFG₁ could decrease the expression of SP-C protein in the cultured AT-II.5 Effects of JNK signaling pathways on cell injury of A549 cells induced by AFG₁Objective: To explore the effects of JNK signaling pathways on the injury of AT-II cell line-A549 cells induced by AFG₁.Methods:5.1 Determination of the effects of AFG₁ on cell injury, activation of JNK, the concentrations of [Ca²⁺]i and the expression of SP-C mRNA in A549 cellsAfter initial culture for 24h, A549 cells were harvestd, centrifuged and resuspended in fresh DMEM medium supplemented with 10% FCS at the concentration of (1²)×10⁸cells·L^-1 in 96-well culture plates. The cells were randomly divided into 6 groups: control, solvent control, AFG₁ 0.1 mg·L^-1, AFG₁ 0.5 mg·L^-1, AFG₁ 1.0 mg·L^-1 and AFG₁2.0mg·L^-1 at each treat time point. The medium of A549 cells was replaced by new DMEM medium supplemented with 2% FCS at 37℃and 5% CO2 24h later. Then the cells in different groups were incubated with different concentrations of AFG₁ (final concentrations of AFG₁ 0.1, 0.5, 1.0 and 2.0mg·L^-1), or DMSO and saline for 8, 16, 24 and 48h. Cell viability for A549 cells was assessed with MTT assay.A549 cells were harvestd, centrifuged and resuspended in DMEM medium supplemented with 10% FCS at the concentration of (1²)×10⁸ cells·L^-1 in culture flasks (8 ml) and 24-well plates. The cells were randomly divided into 5 groups: control, solvent control, AFG₁ 0.5 mg·L^-1, AFG₁ 1.0 mg·L^-1 and AFG₁2.0mg·L^-1. The medium of A549 cells was replaced by new DMEM medium supplemented with 2% FCS 24h later. Then the cells in AFG₁ groups were respectively treated with AFG₁, 0.5 mg·L^-1, 1.0mg·L^-1 and AFG₁2.0mg·L^-1, while these in solvent control and control group were incubated with DMSO and saline respectively. The cells were cultured for 24 h after treatment and harvested for detection. The concentrations of [Ca²⁺]i of A549 cells in 24 well plates loaded with Fluo-3/AM were observed under CLSM. The phospho-JNK (p-JNK) levels of A549 cells treated with AFG₁ were determined by the immunocytochemical stainning and Western blot. The expression of SP-C mRNA of A549 cells was detected by RT-PCR.5.2 Determination of the effects of AFG₁ on cell injury, activation of JNK, and the expression of SP-C mRNA in A549 cells pretreated with SP600125The cells in 96-well culture plates were randomly divided into 9 groups: control, solvent control, JNK inhibitors SP600125 0.1μM, SP600125 0.5μM, SP600125 1μM, 0.1μM SP600125+AFG₁ 1.0mg·L^-1, 0.5μM SP600125+AFG₁ 1.0mg·L^-1, 1μM SP600125+AFG₁ 1.0mg·L^-1 and AFG₁ 1.0mg·L^-1. The medium of A549 cells was replaced by new DMEM medium supplemented with 2% FCS 24h later. The cells of solvent control and control were incubated with DMSO and saline respectively. The cells in other groups (except AFG₁ 1.0mg·L^-1 group) were pretreated for 30 min with different concentrations of SP600125 and then 1.0mg·L^-1 AFG₁ were added into the medium of 0.1μM SP600125+AFG₁ 1.0mg·L^-1, 0.5μM SP600125+AFG₁ 1.0mg·L^-1, 1μM SP600125+AFG₁ 1.0mg·L^-1 and AFG₁ 1.0mg·L^-1 group. Cell viability for A549 cells was assessed with MTT assay after the cells were cultured for another 24h.A549 cells seeded at (1²)×10⁸ cells·L^-1 in culture flasks were randomly divided into 4 groups: control, solvent control, JNK inhibitors and AFG₁ 1.0 mg·L^-1. The medium of A549 cells was replaced by new DMEM medium supplemented with 2% FCS 24h later. The cells of inhibitors group were pretreated for 30 min with 0.5μM SP600125. Then the cells in inhibitors and AFG₁ group were treated with AFG₁ 1.0 mg·L^-1, while these in solvent control and control groups were incubated with DMSO and saline respectively. The cells were cultured for 24 h after treatment and harvested for detection. The JNK activation of A549 cells treated with AFG₁ were determined with Western blot using antibodies specific for p-JNK. The expression of SP-C mRNA of A549 cells was detected by RT-PCR.Results:5.1 Effects of AFG₁ on cell viability for A549 cellsEight hours after treatment, no significant differences were found in the survival rates of A549 cells between all AFG₁ treatment groups and the solvent DMSO group. While 16, 24 and 48h after AFG₁ treatment, the survival rates of A549 cells in AFG₁ treated groups were significantly lower than those in corresponding DMSO groups (p<0.05). The changes in survival rate were not seen in 0.1 mg·L^-1AFG₁ group 16 and 24 h after AFG₁ treatment.5.2 Effects of AFG₁ on the phospho-JNK levels of A549 cellsBlue fluorescence of p-JNK was visualized by CLSM in ecah immunocytochemical stained sample. Fluorescence intensity of p-JNK in 0.5, 1.0 and 2.0 mg·L^-1AFG₁ group was 126±11, 157±14 and 175±11 respectively, which was significantly higher than that in DMSO group (104±9, p<0.05).Western blot results showed that the p-JNK levels in AFG₁ treated A549 cells were increased as compared with that in DMSO group. The relative p-JNK levels in 0.5, 1.0 and 2.0 mg·L^-1 AFG₁ treated groups were 0.40±0.02, 0.51±0.08 and 0.58±0.05 respectively, which was all significantly higher than that in DMSO group (0.29±0.05, p<0.05).The results showed that AFG₁ could cause the activation of JNK in A549 cells.5.3 Effects of AFG₁ on the expression of JNK protein in A549 cellsNo difference in the expression of JNK among the all AFG₁ treated groups and the DMSO group was found by Western blot using antibodies specific for JNK.5.4 Effects of AFG₁ on the concentrations of [Ca²⁺]i of A549 cellsThe concentrations of [Ca²⁺]i in0.5, 1.0 and 2.0 mg? L^-1 AFG₁-treated groups were significantly higher than that in DMSO group(169±13, 204±16 and 228±11 respectively, vs 134±12, p<0.01). A significant dose-depended response correlation could be found between concentrations of [Ca²⁺]_i and AFG₁ concentrations (r=0.932, p<0.01).5.5 Effects of AFG₁ on the expression of SP-C mRNA in A549 cellsThe effects of AFG₁ on the decrease of SP-C mRNA expression were investigated by RT-PCR. The relative expressions of SP-C mRNA in A549 cells in 0.5, 1.0 and 2.0 mg? L^-1 AFG₁ treated groups were 0.39±0.11, 0.30±0.03 and 0.28±0.02 respectively, which were all significantly lower than that in DMSO group (0.56±0.16, p<0.05).5.6 Effects of AFG₁ on the cell survival rates of A549 pretreatment with different concentrations of SP600125The survival rates of 1.0 mg·L^-1 AFG₁ treated A549 cells pretreated with 0.1, 0.5μM SP600 125 and 1.0 mg·L^-1 were 87±5% and 89±5% respectively, which significtantly higher than that in only 1.0 mg·L^-1 AFG₁ treated group 78±8%, but lower than that in DMSO group (98±10%, p<0.05). The results suggest the pretreatment with low concentrations of SP600125 may partly reduce the cell injury of AFG₁ and the activation of JNK pathway in AFG₁-treated cells may partly modulate the AFG₁ related cell injury.5.7 Effects of AFG₁ on the p-JNK levels and SP-C mRNA expression in A549 pretreatment with 0.5μM SP600125Pretreatment of A549 cell with 0.5μM SP600125 could significantly decrease the relative p-JNK level as compared with that of 1.0 mg·L^-1 AFG₁ treatment alone. The effect of activation of JNK in AFG₁-treated cells was inhibited in the presence of SP600125.RT-PCR results showed that there was no difference in the expression of SP-C mRNA in A549 cells between SP600 125 pretreatment group and only 1.0 mg·L^-1 AFG₁ treatment group. The relative expression of SP-C mRNA in P600 125 pretreatment group and 1.0 mg·L^-1 AFG₁ treatment groups was 0.37±0.08 and 0.35±0.05 respectively, which was significant lower than that in DMSO group (0.66±0.11, p<0.05). The presence of SP600125 did not affect the expression of SP-C mRNA in A549 cells treated with AFG₁. The results suggest that activation of the JNK pathway in AFG₁-treated cells was not involved in the modulation of SP-C mRNA.Conclusions:1. The lung adenocarcinomas induced by AFG₁ in NIH mice arised from alveolar type II cells. No positive expression of mutant P53 and Ras P21 was found in the lung adenocarcinomas. Oral administration of AFG₁ for long time could significantly increase the expression of SP-C in AT-II and promote proliferation of alveolar epithelial cells in NIH mice.2. Intratracheal administration of AFG₁ in rats could increase the activities of LDH and AKP in BALF and cause ultrastructural injury changes of lung tissues in acute stage. AFG₁ could increase the expression of TNF-αmRNA in alveolar cells, decrease the antioxidant ability but have on effect on the expression of NF-κB in rat lung tissues.3. Intratracheal administration of AFG₁ in rats could cause ultrastructural injury changes of AT-II and decrease the expression of SP-C and SP-A mRNA in lung tissues.4. AFG₁ could cause injuries in cultured rat lung alveolar type-â…¡cells. At the same time, AFG₁ could increase the concentration of [Ca²⁺]i and decrease the expression of SP-C in AT-II in vitro.5. AFG₁ could elevate the concentration of [Ca²⁺]i, cause the activation of JNK and decrease the expression of SP-C mRNA in A549 cells. Pretreatment of JNK inhibitor, SP600125 may partly reduce the cell injury by AFG₁ but have no effects on the expression of SP-C mRNA. The activation of the JNK pathway in AFG₁-treated cells may partly modulate the cell injury.