Methyl methacrylate toxicity in rat nasal epithelium: studies of the mechanism of action and comparisons between species.

Female F344 rats exposed to 200 ppm methyl methacrylate for 6 h developed a lesion in the nasal olfactory epithelium which was characterised by degeneration and atrophy. The severity of the lesion was markedly reduced by pre-treatment of the rats with an intraperitoneal dose of 100 mg/kg bis-(p-nitrophenyl)phosphate, an inhibitor of carboxylesterase enzymes, thus demonstrating that the lesion is caused by the carboxylesterase mediated metabolism of methyl methacrylate to methacrylic acid, an irritant and corrosive metabolite. The distribution of the carboxylesterases in nasal tissues has been investigated and the metabolism of methyl methacrylate to methacrylic acid has been compared in rat, hamster and human nasal tissue fractions in vitro. Histocytochemistry showed that the carboxylesterases are heavily localised in the sustentacular cells and Bowman's glands of the rat olfactory region, but are more generally distributed in human olfactory epithelium. Consistent with this, the enzyme activity in all three species was higher in fractions prepared from olfactory tissue than from respiratory tissue, 3-fold in rat and human and 12-fold in the hamster. The maximum rates (V(max)) of metabolism in rat and hamster olfactory tissue fractions were comparable, whereas those in human olfactory tissue fractions were at least 13-fold lower. The rate of metabolism in rat olfactory tissue was also comparable to that in rat liver whereas in humans, the rate in olfactory tissue was 500-fold lower than that in the liver. In respiratory tissues, the rate in humans was at least 6-fold lower than that in the rat. These results suggest that humans are significantly less sensitive than rodents to the nasal toxicity of methyl methacrylate.

Trichloroethylene-induced mouse lung tumors: studies of the mode of action and comparisons between species.

CD-1 mice exposed to 450 ppm trichloroethylene, 6 hr/day, 5 days/week, for 2 weeks showed a marked vacuolation of lung Clara cells after the first exposure of each week and a marked increase in cell division after the last exposure of each week. The damage seen in mouse lung Clara cells is caused by an accumulation of chloral resulting from high rates of metabolism of trichloroethylene but poor clearance of chloral to trichloroethanol and its glucuronide. The activity and distribution of the key metabolizing enzymes in this pathway have been compared in mouse, rat, and human lung. While mouse lung microsomal fractions were able to metabolize trichloroethylene to chloral at significant rates, the rate in rat lung was 23-fold lower and a rate could not be detected in human lung microsomes at all. Immunolocalization of cytochrome P450IIE1 in lung sections revealed high concentrations in mouse lung Clara cells with lesser amounts in type II cells. Lower levels of enzyme could be detected in Clara cells of rat lung, but not at all in human lung sections. Western blots of lung tissues from the three species and of mouse lung Clara cells were entirely consistent with these observations. Consequently, it is highly unlikely that humans exposed to trichloroethylene are at risk from the lung damage/cell proliferation mechanism that is believed to lead to the development of tumors in the mouse lung.

The distribution of theta-class glutathione S-transferases in the liver and lung of mouse, rat and human.

Two murine Theta-class glutathione S-transferases (GSTs), mGSTT1 and mGSTT2, have been cloned and sequenced. The murine cDNAs, together with the published sequences of the rat and human enzymes, were used to design oligonucleotide probes in order to determine the distribution of mRNA for these enzymes in the liver and lung of rat, mouse and human. The mRNA distribution was compared with that of enzyme protein determined with an antibody to rat GSTT2-2. Both the antibody and the oligonucleotide probes gave the same distribution patterns. Both enzymes were present at significantly higher concentrations in mouse tissues than in rat or human tissues. In mouse liver, both enzymes were localized in specific cell types and in nuclei. Although the distribution of GSTT2-2 in rat liver was similar to that seen in the mouse, GSTT1-1 was not localized in a specific cell type or in the nuclei of either rat or human liver. In the lungs, very high concentrations of the Theta enzymes were present in mouse-lung Clara cells and ciliated cells, with much lower levels in the Clara cells only of rat lung. Low levels of human transferase GSTT1-1 were detected in a small number of Clara cells and ciliated cells at the alveolar/ bronchiolar junction. The relative activities between species, and the cellular and sub-cellular distribution within the liver and lungs of each species, provides an explanation for the species-specificity of methylene chloride, a mouse-specific carcinogen activated by glutathione S-transferase GSTT1-1.

Isolation of a mouse theta glutathione S-transferase active with methylene chloride.

A glutathione S-transferase metabolizing methylene chloride has been isolated from mouse liver using a variety of chromatographic methods. N-terminal and internal amino acid sequences show that the enzyme, designated GST T1-1*, is closely related to the rat Theta-class GST 5-5. The mouse enzyme, molecular mass 25000 Da, has been isolated to homogeneity in active form with an approximate yield of 2% of the cytosolic activity towards methylene chloride. GST T1-1* has a specific activity of about 5.5 micromol/min per mg of protein whereas the rat GST 5-5 is reported to have a specific activity of about 11 micromol/min per mg of protein [Meyer, Coles, Pemble, Gilmore, Fraser and Ketterer (1991) Biochem. J. 274, 409-414], demonstrating that both the rat and mouse enzymes have similar activity with this substrate. Limited evidence was obtained for a second enzyme, with a similar molecular mass (25400 Da), which had an N-terminal sequence identical to that of rat GST 12-12. This protein, which was sequenced from a band on a gel, was extremely labile and could not be isolated to homogeneity. The partially purified enzyme was not active with methylene chloride.

Glutathione depletion in the liver and brain produced by 2-chloropropionic acid: relevance to cerebellar granule cell necrosis.

L- and D-2-chloropropionic acid (L-CPA and D-CPA) produce selective damage to granule cells of the rat cerebellum by a mechanism that is not currently understood. We have demonstrated that both L- and D-CPA produce a rapid, dose and time dependent depletion of liver non-protein sulphydryl (NP-SH) content, mainly glutathione (GSH), while in the cerebellum and forebrain, there is a slower, dose and time dependent decrease in NP-SH. Twenty-four hours after a single dose of 750 mg/kg L-CPA (a dose sufficient to produce cerebellar toxicity, but a time prior to the onset of cellular necrosis), the content of total GSH was depleted by 85% in the cerebellum and to a lesser degree in the forebrain, while no increase in oxidised glutathione was observed in either tissue. In vitro both L- and D-CPA caused a marked reduction in GSH concentration when incubated with hepatic cytosol but not hepatic microsomes or brain cytosol. The hepatic cytosolic depletion appeared to be due to a direct reaction catalysed by a theta class glutathione S-transferase. A GSH adduct of L-CPA was isolated by high pressure liquid chromatography and identified by mass spectrometry as 2-S-glutathionyl propanoic acid, confirming a direct substitution reaction. No GSH adducts were formed by cerebellar or forebrain cytosol, suggesting that the particular isoform of glutathione S-transferase catalysing the reaction may not be present in the brain. We suggest that the marked and sustained CPA-mediated GSH depletion in the granule cells of the cerebellum may render these cells more vulnerable to oxidative free radical damage.

The effect of anticoagulants on Blennius pholis L. Leucocytes.

The effects of ethylenediamine-tetraacetic acid (EDTA), heparin and tri-sodium citrate (TSC) on various haematological parameters in the blenny, Blennius pholis, were investigated. EDTA and heparin, at the concentrations tested, proved adequate in preventing coagulation although leucocyte viability was reduced when the higher levels were used. TSC failed to prevent coagulation at all concentrations tested. Short (5 min) and long (20 min) term contact of these anticoagulants with blood did not produce any significant changes in leucocyte proportions. Heparin is regarded as the most suitable anticoagulant for use with B. pholis blood.

Separation of leucocytes in the dogfish (Scyliorhinus canicula) using density gradient centrifugation and differential adhesion to glass coverslips.

The blood of the dogfish, S. canicula, contains several types of leucocytes, namely thrombocytes, monocytes, lymphocytes and four populations of granulocytes. Three of these granulocyte types, G1, G3 and G4, are eosinophilic while G2 is heterophilic/neutrophilic. All of the leucocyte types, with the exception of G2 granulocytes and monocytes, can be separated by means of their differential adherent properties to glass and by density gradient centrifugation. Thrombocytes, G3 and G4 granulocytes can be separated in good purity by single-step methods while G1 granulocytes and lymphocytes require a combination of density gradient centrifugation followed by adherence to glass to remove contaminating thrombocytes. Depending on the cell type, between 11-45% of cells with consistently high viability can be recovered after separation. Separated populations of the thrombocytes and granulocytes will be especially useful for studies on the role of such cell types in inflammation.