Cytotoxicity and stress gene microarray analysis in cadmium-exposed CRL-1439 normal rat liver cells.

Cadmium is a biologically non-essential divalent hazardous metal. Previous studies demonstrated that cadmium toxic effect was caused by reactive oxygen species. Since gene expression is influenced by the presence of these reactive oxygen species, the association between metal intoxication and gene expression has recently become a major focus of research. We examined the effect of cadmium chloride on cell viability at 4, 8 and 24 h. Our results indicate that cadmium chloride did not alter cell viability at 4 or 8 h, but decreased the viability in a dose-dependent manner (p>0.01) at 24 h. Using DNA microarray, we studied the profile of stress gene expression in rat primary hepatocytes treated with cadmium for different time periods using a 100 microM cadmium chloride concentration. Microarray analysis indicated that cadmium treatment caused different patterns of gene expression profiles at each time point of incubation. Of the 207 stress genes on the microarray, only 32 genes were regulated. Since microarrays were hybridized by radioactive cDNA which was less sensitive than fluorescent-labeled cDNA, an experimental/control ratio >1.3 or <0.7 (30% increase or decrease) was taken as significant up- or down-regulation. Exposure of cells to cadmium for 4 h resulted in the expression of three up-regulated genes and six down-regulated genes. Longer exposure to cadmium for 8 h resulted in an increase in up-regulated genes to six and down-regulated genes to 14. After 24 h of cadmium exposure, 15 genes were down-regulated and six genes were up-regulated. Our findings suggest that the cells maintained complete viability up to 8 h with cadmium due to expression of various heat shock proteins and stress response proteins like heme oxygenase. Longer exposure periods, due to the down-regulation of the basic cell function proteins and cell-cycle regulating proteins, led to toxicity in cells and eventually to cell death.

Comparative evaluation of cytotoxicity of cadmium in rat liver cells cultured in serum-containing medium and commercially available serum-free medium.

Cadmium (Cd) is an industrial pollutant and carcinogenic metal. Most in vitro Cd toxicity studies have been carried out in various cell lines cultured in 10% fetal bovine serum (FBS) containing medium. In this report, we compared the toxic effect of Cd (0-300 microM) on cell growth, total RNA, total proteins, and antioxidant enzymes in rat normal liver cells cultured in medium with 10% FBS or commercially available serum-free medium for 4 or 8 hours. With Cd concentration at above 100 microM, the total levels of RNA, protein and cell growth decreased in serum-containing medium, while their levels increased in serum-free medium compared to the controls. The glutathione peroxidase and glutathione reductase levels were lower in serum-free medium than in serum-containing medium, indicating less oxidative stress in cells grown in serum-free medium. These results clearly suggest that Cd showed higher toxicity to liver cells grown in serum-containing medium in comparison to commercially available serum-free medium. It is speculated that albumin and other substances present in commercial serum-free medium chelated Cd and thereby protected these cells against Cd toxicity. Even under in vivo conditions, cadmium enters into various organs after passing through blood which contains serum. Based on these studies, it appears that media containing serum may be ideal for in vivo toxicity correlation studies with animal cells.

Role of pneumococcal surface protein C in nasopharyngeal carriage and pneumonia and its ability to elicit protection against carriage of Streptococcus pneumoniae.

Previous studies suggested that PspC is important in adherence and colonization within the nasopharynx. In this study, we conducted mutational studies to further identify the role PspC plays in the pathogenesis of pneumococci. pspC and/or pspA was insertionally inactivated in a serotype 2 Streptococcus pneumoniae strain and in a serotype 19 S. pneumoniae strain. In the mouse colonization model, pneumococcal strains with mutations in pspC were significantly attenuated in their abilities to colonize. In a mouse pneumonia model, strains with mutations in pspC were unable to infect or multiply within the lung. Using reverse transcriptase PCR we were able to demonstrate that pspC is actively transcribed in vivo, when the bacteria are growing in the nasal cavity and in the lungs. In the bacteremia model, a strain mutated for pspC alone behaved like the wild type, but the absence of both pspC and pspA caused accelerated clearance of the bacteria. Intranasal immunization with PspC with cholera toxin subunit B as an adjuvant protected against intranasal challenge. Evidence was also obtained that revertants that spontaneously acquired PspC expression could multiply and colonize the nasal tissue. This latter finding strongly indicates that pneumococci are actively metabolizing and growing while in the nasopharynx.