Characterizing the mechanism of thiazolidinedione-induced hepatotoxicity: An in vitro model in mitochondria.

OBJECTIVE: To characterize the mechanism of action of thiazolidinedione (TZD)-induced liver mitochondrial toxicity caused by troglitazone, rosiglitazone, and pioglitazone in HepaRG cells. METHODS: Human hepatoma cells (HepaRG) were treated with troglitazone, rosiglitazone, or pioglitazone (12.5, 25, and 50µM) for 48h. The Seahorse Biosciences XF24 Flux Analyzer was used to measure mitochondrial oxygen consumption. The effect of TZDs on reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected by flow cytometry. The mitochondrial ultrastructure of HepaRG cells was observed under a transmission electrical microscope (TEM). mtDNA content was evaluated by real-time PCR, and ATP content and mitochondrial respiratory chain (MRC) complex I, II, III, IV activity were measured via chemiluminescence. Results were considered statistically significant at p<0.05. RESULTS: Among the three drugs, troglitazone exhibited the highest potency, followed by rosiglitazone, and then pioglitazone. The TZDs caused varying degrees of mitochondrial respiratory function disorders including decreases in oxygen consumption, MRC activity, and ATP level, and an elevation in ROS level. TZD treatment resulted in mtDNA content decline, reduction in MMP, and alterations of mitochondrial structure. CONCLUSION: All investigated TZDs show a certain degree of mitochondrial toxicity, with troglitazone exhibiting the highest potency. The underlying mechanism of TZD-induced hepatotoxicity may be associated with alterations in mitochondrial respiratory function disorders, oxidative stress, and changes in membrane permeability. These parameters may be used early in drug development to further optimize risk:benefit profiles.

[Protective effect of insulin-like growth factor-1 on acute lung injury induced by perfluoroisobutylene inhalation in mice].

OBJECTIVE: To observe the protective effect of insulin-like growth factor-1 (IGF-1) on acute lung injury induced by perfluoroisobutylene (PFIB) inhalation in mice. METHODS: Sixty-four male Kunming mice were randomly divided into normal control (A) group, exposed (B) group, recombinant adenoviruses 5 of IGF-1 (Ad5-IGF-1) intervention (C) group (in which Ad5-IGF-1 was injected into the trachea of the mice), blank vector control (D) group. B, C and D groups were exposed to gaseous PFIB in a flow-past whole-body exposure system. The lung index, concentration of total protein and albumin in bronchoalveolar lavage fluid (BALF), concentration of IGF-1 in serum and lung homogenate were measured. The lung pathologic changes were examined with light microscope, and ultrastructure changes in alveolar type II cells (ATII) with electron microscope. RESULTS: Compared with A group, the lung index, concentration of total protein in BALF were significantly increased in other groups, the lung index and concentration of total protein and albumin of BALF in B and D groups were prominently higher than C group (all P<0.01). The concentration of IGF-1 in serum of B and D groups was lower markedly than that of A group, and the concentration of IGF-1 in serum of C group was distinctly higher than those of A, B, D groups (all P<0.01). The concentration of IGF-1 in lung homogenate of B, C, D groups was higher than that of A group, and the concentration of IGF-1 in lung homogenate of C group was significantly higher than that of B and D groups (all P<0.01). Lung hyaline membrane formation, diffuse alveolar atelectasis, accumulation of edema fluid, red blood cell exudation, were obviously milder in C group, and changes in the ultrastructure of ATII showed a similar result. CONCLUSION: The protective effect of Ad5-IGF-1 against the toxicity of PFIB inhalation is identified. In the mice pretreated with Ad5-IGF-1 is able to significantly lower lung index, the protein concentration in BALF, and the concentration of IGF-1 in serum and lung homogenate is obviously increased. Protection of ATII may be one of the mechanisms.

A simple method for the formalin fixation of lungs in toxicological pathology studies.

Optimized lung preparation for detailed structural evaluation is required to improve consistency in preclinical safety evaluation, differences of opinion exist among regulatory agency personnel regarding the optimal methods for routine formalin fixation of lungs from rodent toxicology studies. The simple tracheal ligation fixation method emphasizes tracheal ligation before opening the thorax instead of attempting to re-inflate after lung collapse when opening the thorax. Photomicrographs of this method demonstrated an unprecedented ability to maintain the natural lung architecture, in contrast to the unavoidable changes in the alveolar environment by the intratracheal instillation and vascular perfusion methods. In addition, a comparison of fixation methods on lung morphology in a rodent model of LPS-induced acute lung injury demonstrated that the tracheal ligation fixation method may provide a standard approach for morphometry. Additionally, a TUNEL assay was used to determine the degree of autolysis, which revealed that the autolysis was insignificant in the central areas of each lobe of the lung compared to the lung periphery by tracheal ligation fixation. In conclusion, our novel modified method, which avoids the disadvantages of generating artifacts, fulfills the requirement of preserving the clear, natural morphology of the lung making it suitable and worthy of recommendation for toxicological studies in a good laboratory practice (GLP) lab.

[The clinical efficacy of hemoperfusion in treatment of drug poisoning].

OBJECTIVE: To investigate the ability of hemoperfusion to remove some drugs or toxin from the body and its clinical efficacy. METHODS: Sixty-nine cases of poisoning due to tetramine or other drugs poisoning in our hospital between July 1990 and December 2003 were studied. Thirty-four patients among them received conventional treatment (including early gastric lavage, hepatoprotection, diuresis or respiratory support), and the remaining were given conventional treatment and hemoperfusion. Toxin concentration changes in blood before and after hemoperfusion, survival rate, time from come onset to regaining consciousness and convulsion termination and duration of clinical course were compared. RESULTS: In the hemoperfusion group, three patients died, the survival rate was 91.4%, whereas in the non-hemoperfusion group, the survival rate was 85.3% (P > 0.05). Meanwhile the clinical course was markedly shortened in the hemoperfusion group (P < 0.05). The time from coma onset to regaining consciousness and convulsion termination in the hemoperfusion group was significantly shorter than that in the non-hemoperfusion group. The clearance rate was different for different poisons, among them the benzodiazepines had an excellent clearance. CONCLUSIONS: Hemoperfusion could adsorb from blood a different amount of poisons. There is obvious efficacy in shortening clinical course and reducing complications. It could possibly raise survival rate in serious poisoning.

[Modeling of acute respiratory distress syndrome in canine after inhalation of perfluoroisobutylene and preliminary study on mechanisms of injury].

OBJECTIVE: To establish of acute respiratory distress syndrome (ARDS) model in canine after inhalation of perfluoroisobutylene (PFIB), and to observe the progressing of lung injury, and to study the mechanisms of injury. METHODS: A device of inhalation of PFIB for canine was made. The concentration of PFIB was 0.30 - 0.32 mg/L. Serum IL-6 and IL-8 were dynamically measured. Clinical manifestations, pathology of organs in canine were observed. RESULTS: (1) During inhalation, the concentration of PFIB remained stable; (2) After inhalation, blood arterial oxygen partial pressure fell gradually, and eventually met the criteria for diagnosing ARDS; (3) The level of IL-8 in serum rises significantly after inhalation (P < 0.05), whereas that of IL-6 was not obviously altered (P > 0.05); (4) Within 6 hours after inhalation, no abnormality in canine was observed, but afterwards symptoms gradually appeared, and typical breath of ARDS, such as high frequency and lower level could be seen in later phase; (5) Pathological examination showed severe congestion, edema and atelectasis in most part of both lungs, and signs of anoxia in other organs. CONCLUSIONS: (1) The device designed is capable of ensuring control of inhalation of PFIB; (2) Exposure to PFIB for 30 mins, canines all met the criteria for diagnosing ARDS 22 hours after inhalation, therefore the modeling is successful; (3) PFIB specifically damages the lung by causing excessive inflammation.

Subchronic toxicological study of two artemisinin derivatives in dogs.

The objective of our study was to profile and compare the systematic changes between orally administered artesunate and intramuscularly injected artemether at a low dose over a 3-month period (92 consecutive days) in dogs. Intramuscular administration of 6 mg kg-1 artemether induced a decreased red blood cell (RBC) count (anemia), concurrent extramedullary hematopoiesis in the spleen and inhibition of erythropoiesis in the bone marrow. We also observed a prolonged QT interval and neuropathic changes in the central nervous system, which demonstrated the cortex and motor neuron vulnerability, but no behavioral changes. Following treatment with artesunate, we observed a decreased heart rate, which was most likely due to cardiac conduction system damage, as well as a deceased RBC count, extramedullary hematopoiesis in the spleen and inhibition of erythropoiesis in the bone marrow. However, in contrast to treatment with artemether, neurotoxicity was not observed following treatment with artesunate. In addition, ultra-structural examination by transmission electron microscopy showed mitochondrial damage following treatment with artesunate. These findings demonstrated the spectrum of toxic changes that result upon treatment with artesunate and artemether and show that the prolonged administration of low doses of these derivatives result in diverse toxicity profiles.

Injury of cell tight junctions and changes of actin level in acute lung injury caused by the perfluoroisobutylene exposure and the role of Myosin light chain kinase.

OBJECTIVES: To investigate the injury of cell tight junctions and change in actin level in the alveolus epithelial cells of the lung after perfluoroisobutylene (PFIB) exposure and the role of myosin light chain kinase (MLCK) in the injury. METHODS: Rats and mice were exposed to a sublethal dose of PFIB. The changes in tight junction zonula occludens-1 (ZO-1), actin and myosin light chain kinase (MLCK) were detected by immunofluorescence at 30 min, 1, 2, 4, 8, 16, 24, 48 and 72 h after PFIB exposure. The role of MLCK was analyzed by lung indices and the actin level. RESULTS: The normal ZO-1 immunofluorescence density and those after PFIB exposure were 71.63, 39.41, 37.59, 35.71, 33.22, 31.34, 31.61, 24.51, 40.03 and 44.71 respectively, The normal actin immunofluorescence density and those after PFIB exposure were 31.82, 36.46, 36.57, 41.60, 40.95, 35.41, 30.69, 19.96, 29.30 and 33.00 respectively, The normal MLCK immunofluorescence density and those after PFIB exposure were 61.21, 50.87, 48.37, 43.65, 41.96, 35.44, 31.77, 30.85, 33.10 and 38.20 respectively. When the MLCK inhibitor ML-7 was given in advance, pulmonary edema and actin degradation were suppressed. CONCLUSIONS: At an earlier stage, the increased permeability of the blood-air barrier after PFIB exposure is probably the result of injury of cell tight junctions that acts in concert with later changes in actin, resulting in an increase in permeability. MLCK could be a potential target for novel drug development for relief of acute lung injury.

Cell injuries of the blood-air barrier in acute lung injury caused by perfluoroisobutylene exposure.

OBJECTIVES: To investigate the complete process of cell injuries in the blood-air barrier after perfluoroisobutylene (PFIB) exposure. METHODS: Rats were exposed to PFIB (140 mg/m(3)) for 5 min. The pathological changes were evaluated by lung wet-to-dry weight ratio, total protein concentration of bronchoalveolar lavage fluid and HE stain. Ultrastructural changes were observed by transmission electron microscope. Apoptosis was detected by in situ apoptosis detection. Changes of actin in the lung tissue were evaluated by western blot assay. RESULTS: No significant pulmonary edema or increased permeability was observed within the first 4 h, post PFIB exposure. However, inflammatory cell infiltration and alveolar wall thickening were observed from 2 h. Destruction of the alveoli constitution integrity, edema and protein leakage were observed at 8 h. The injuries culminated at 24 h and then recovered gradually. The ultrastructural injuries of alveolar type I epithelial cells, alveolar type II epithelial cells and pulmonary microvascular endothelial cells were observed at 30 min post PFIB exposure. Some injuries were similar to apoptosis. Compared with control, more serious injuries were observed in PFIB-exposed rats after 30 min. At 8 h, some signs of cell necrosis were observed. The injuries culminated at 24 h and then ameliorated. The number of apoptotic cells abnormally increased at 30 min post PFIB exposure, the maximum appeared at 24 h, and then ameliorated gradually. Western blot analysis revealed that the level of actin in the lung showed no significant changes within the first 4 h post PFIB exposure. However, it decreased at 8 h, reached a nadir at 24 h, and then recovered gradually. CONCLUSIONS: The pathological processes were in progress persistently post PFIB exposure. The early injuries probably were the result of the direct attack of PFIB and the advanced injuries probably arose from the inflammatory reaction induced by PFIB.