Novel and extensive aspects of paraquat-induced pulmonary fibrogenesis: comparative and time-course microarray analyses in fibrogenic and non-fibrogenic rats.

Although paraquat (PQ) is widely known to induce pulmonary fibrosis, the molecular mechanisms are poorly understood. Therefore, to bring a new dimension to the elucidation of the mechanisms, we conducted microarray experiments to investigate the expression profiles of 1,090 genes in the lungs during the progressive phase of PQ-induced pulmonary fibrosis in rats. After several s.c. injections of PQ, rats were divided into a fibrogenic group and a non-fibrogenic group. Time-course gene expression analysis of the fibrogenic group showed altered gene regulation throughout the experimental period. The expression levels of many cell membrane channel, transporter, and receptor genes were substantially altered. These genes were classified into two categories: polyamine transporter- and electrolyte/fluid balance-related genes. Moreover, comparative analysis of the fibrogenic and the non-fibrogenic group revealed 36 genes with significantly different patterns of expression, including the pro-apoptotic gene Bad. This indicates that Bad is a key factor in apoptosis and that apoptosis provides a major turning point in PQ-induced pulmonary fibrosis. Notably, subtypes of transforming growth factor (TGF)-beta genes that are considered to play a pivotal role in fibrogenesis showed no differences in expression between the two groups, though TGF-beta3 was markedly induced in both groups. These results provide novel and extensive insights into the molecular mechanisms that lead to pulmonary fibrosis after exposure to PQ.

DNA microarray analysis of pulmonary fibrosis three months after exposure to paraquat in rats.

Although paraquat (PQ) is known to induce pulmonary fibrosis, how it does so is not entirely clear. To elucidate the mechanisms involved, the profile of gene expression in the lung at three months after exposure to PQ (7 mg/kg, s.c., daily for eight administrations) was investigated in rats using a DNA microarray. Changes in gene expression that were considered to reflect damage to the lung, a change in the balance of electrolytes and fluid, and alveolar remodeling were observed. The products of these genes were: CSF-1 receptor, which is a receptor of inflammatory cytokines that activates monocyte/macrophages; TGF-beta type II receptor, which is a receptor of TGF-betas involved in wound healing and fibrosis; a subunit of Na+/K(+)-ATPase, an amiloride-sensitive cation channel, and a subunit of the potassium channel, all of which regulate the alveolar fluid balance and play a role in clearing lung edema; the adenosine A2a receptor, which has a protective function in the lung and interacts with dopamine D1 and D2 receptors to regulate the function of amiloride-sensitive cation channels; cofilin, which is involved in the depolymerization and cleavage of actin filaments; LIM motif-containing protein kinase 1, which negatively regulates the activity of cofilin; SHPS-1, which regulates the integrin-mediated reorganization of the cytoskeleton; and sodium channel beta 2, which is involved in cell adhesion and migration. These results indicate that PQ-induced pulmonary fibrosis does not merely terminate as cicatrices three months after the discontinuation of PQ treatment, but that dynamic functional change continues in the lung.

Flow cytometric analysis of erythropoietic abnormality: changes in the cell maturity index of reticulocytes and retic distribution index are useful as indicators of erythropoietic toxicity in non-clinical studies.

We performed a flow cytometric (FCM) analysis of the maturity of reticulocytes using peripheral blood obtained from rats administered 5-fluorouracil (5-FU) at 10, 50 and 100 mg/kg or acethylphenyl hydrazine (APHZ) at 1 and 3 mg/kg to clarify whether the FCM method is useful for assessing toxicity. In the 5-FU-administered rats, a decrease and recovery of the immature reticulocyte fraction (Cell Maturity Index, CMI; Retic Distribution Index, RDI) was observed more rapidly (several days prior to changes in the reticulocyte ratio), and sensitively regarding dose-dependency (clear changes were observed at 10 mg/kg, whereas the reticulocyte ratio was only slightly affected). In addition, there was good agreement between the microscopic results obtained by counting Heilmyer's reticulocyte maturation groups, especially for type I and II, and CMI/RDI assessed by the FCM method after the administration of 50 and 100 mg/kg of 5-FU, the dose at which clear changes were obtained with the microscopic method. In the APHZ-administered rats, a dose-dependent increase in CMI/RDI coinciding with the enhancement of reticulocyte production was observed. The results suggested that the automated FCM method could be a useful and valuable tool to assess and predict impairments of erythropoiesis, especially for CMI and RDI, and could help in the diagnosis of hematological disorders in experimental animals.

Gene expression analysis of the lung following paraquat administration in rats using DNA microarray.

Gene expression changes in the lungs induced by paraquat (PQ) administration were studied in rats using DNA microarrays that were detectable for 1,090 genes per DNA microarray. The rats were subjected to subacute PQ exposure (7 mg/kg, s.c., daily for eight administrations). Two days after the final administration, the rats were divided into two groups. Group 1 experienced significant body weight loss and displayed signs of subacute PQ toxicity, but Group 2 showed no significant effects due to the PQ treatment. A control group, Group 3, was also included. In the comparison of the gene expression levels in the animals from Group 1 or Group 2 to the control animals treated by vehicle, 48 genes in Group 1 and 29 genes from Group 2 were differentially expressed. The twenty-eight genes were common to these two groups. These differentially expressed genes following paraquat treatment were classified as follows: 5 neurotransmitter receptor genes; 4 transporter genes; 4 voltage-gated ion channel genes; 2 lipid metabolism enzyme genes; 2 G-proteins involved in endocytosis and exocytosis genes; 7 cytokine genes; 4 ADP ribosylation genes involved in cell death and regeneration; CFTR gene, which is the causal gene for cystic fibrosis; neurofibromatosis type 1 gene, which is the causal gene for the neurofibromatosis type 1 that is known to accompany pulmonary fibrosis; and the causal gene for spinocerebellar ataxia. These genes may prove to be the keys for the elucidation of the mechanism of PQ toxicity, e.g. PQ-induced pulmonary fibrosis.