Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice.

Mice deleted for the plasminogen activator inhibitor-1 (PAI-1) gene are relatively protected from developing pulmonary fibrosis induced by bleomycin. We hypothesized that PAI-1 deficiency reduces fibrosis by promoting plasminogen activation and accelerating the clearance of fibrin matrices that accumulate within the damaged lung. In support of this hypothesis, we found that the lungs of PAI-1(-/-) mice accumulated less fibrin after injury than wild-type mice, due in part to enhanced fibrinolytic activity. To further substantiate the importance of fibrin removal as the mechanism by which PAI-1 deficiency limited bleomycin-induced fibrosis, bleomycin was administered to mice deficient in the gene for the Aalpha-chain of fibrinogen (fib). Contrary to our expectation, fib(-/-) mice developed pulmonary fibrosis to a degree similar to fib(+/-) littermate controls, which have a plasma fibrinogen level that is 70% of that of wild-type mice. Although elimination of fibrin from the lung was not in itself protective, the beneficial effect of PAI-1 deficiency was still associated with proteolytic activity of the plasminogen activation system. In particular, inhibition of plasmin activation and/or activity by tranexamic acid reversed both the accelerated fibrin clearance and the protective effect of PAI-1 deficiency. We conclude that protection from fibrosis by PAI-1 deficiency is dependent upon increased proteolytic activity of the plasminogen activation system; however, complete removal of fibrin is not sufficient to protect the lung.

Alkaline single-cell gel (comet) assay and genotoxicity monitoring using two species of tadpoles.

Small bodies of water (e.g., creeks, ponds, and drainage ditches) have received very little attention in genotoxicity studies, yet these areas are important because they are often the first to be affected by industrial effluents, sewage contaminants, accidental spills, internal combustion engine emissions, landfill runoffs, and pesticide uses. To address this deficiency, we examined erythrocytes in two species of tadpoles, Rana clamitans and Bufo americanus, using the alkaline single-cell gel (SCG) ("comet") assay. This approach involves detection, under alkaline conditions, of cell DNA fragments, which on electrophoresis migrate from the nuclear core, resulting in a "comet-with-tail" formation. Exposure of R. clamitans todpoles to a range of concentrations of methyl methanesulfonate (MMS) produced a linear increase in DNA length to DNA core width ratios. This is consistent with findings in a number of other species. Time-dose experiments using MMS suggest that the peak level of DNA damage in R. clamitans todpoles occurred 42 hr after exposure. B. americanus tadpoles exposed to 6.25 mg/l of MMS for 12 hours had a significant increase in DNA damage over that seen in the controls. Freshly caught R. clamitans tadpoles from Highgate and B. americanus tadpoles from Duart, both on the north shore of Lake Erie, gave ratios of 2.78 and 2.07, respectively. This region of Ontario is a prime agricultural area and pesticide use is extensive. Tadpoles from Highgate and Duart, maintained in the laboratory for 4 months and 6 weeks, respectively, gave ratios of 1.29 and 1.44. The results of the SCG procedure in tadpoles indicate that this assay is extremely sensitive and suitable for detecting genotoxicity in the environment.

Alkaline single cell gel (comet) assay and genotoxicity monitoring using bullheads and carp.

Monitoring genotoxicity of the environment using endemic organisms as sentinels requires the development of sensitive assays. Toward this end, we explored the feasibility of applying the alkaline single cell gel (SCG) or "comet" assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which, on electrophoresis, migrate from the nuclear core, resulting in a "comet with tail" formation. Tail length has been correlated with level of genotoxicant exposure in a number of organisms. The fish used in this study were benthic feeding bullheads (Ameiurus nebulosus) and carp (Cyprinus carpio). On electrophoresis of erythrocyte DNA under alkaline conditions, we found a linear increase in the tail length/core width ratio over a broad range of cyclophosphamide doses. Freshly caught bullheads from seven different sites showed a wide range of DNA damage. Bullheads from Big Creek (western Lake Erie), Hamilton Harbour (western Lake Ontario), and the Detroit River gave ratios of 3.81 to 4.65. Based on polycyclic aromatic hydrocarbon (PAH) and polychlorinated biphenyl (PCB) levels, the sediment at these three sites is considered to be heavily polluted. Bullheads from southern Lake Huron, which is relatively clean, and from a fish hatchery in Brockport, New York, gave ratios between 1.30 and 1.40. Bullheads from Big Creek, maintained in the laboratory for 3 months, gave ratios which approached those seen in hatchery-bred fish. Results for carp were similar. Carp from Big Creek gave ratios of about 4.50, while carp from Lake Huron and laboratory-maintained carp gave values of 1.23 and 1.36, respectively. The results of the SCG procedure in bullheads and carp indicate that this assay is extremely sensitive and should be useful in detecting DNA damage caused by environmental contaminants.

Induction of mutations by N-acetoxy-N-acetyl-2-aminofluorene modified M13 viral DNA.

The specificity of N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (G-8-AAF) adducts in double-stranded DNAs from M13mp8 and M13mp9 bacteriophage was determined following transfection of modified DNA with multiple adducts into competent JM103 cells. Mutant phages were selected by phenotypic screening for colorless or light blue plaques indicating a defective beta-galactosidase marker enzyme. Mutation frequencies of phage DNA with G-8-AAF adducts were increased up to 8-fold in SOS-induced host cells as compared to the uninduced JM103 host cells. DNA sequencing of mutants from SOS-induced host cells indicated approximately 52% frameshifts and 39% base substitutions in M13mp8 DNA and 65% frameshifts and 25% base substitutions in M13mp9 DNA. Mutation spectra exhibited mutations at many sites within the bp 6200-6400 region; one mutational hotspot at position 6343-6347 (5' GGGGG 3') for frameshifts was also observed. The G-8-AAF adduct induced mostly single base deletions at this site. In contrast, a deacetylated adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene (G-8-AF) in our previous experiments induced mostly single base additions at the same position indicating the ability of adduct structure to modulate the specificity of frameshift mutations. A number of other frameshift mutations (11 out of 29) were observed within non-repetitive and non-palindromic sequences. Molecular mechanisms for the induction of these mutations by DNA perturbations produced by the G-8-AAF adducts are discussed.