The accumulation and metabolism of 3-trifluoromethylpyridine by rat olfactory and hepatic tissues.

The accumulation of [methyl-14C]3-trifluoromethylpyridine (14C-3-FMP) by rat olfactory and hepatic tissue in vivo and in vitro has been investigated. 14C-3-FMP accumulates rapidly and selectively in both tissues in vivo, with an appreciable proportion of this activity being associated with the protein macromolecular fractions. Similar results were seen when isolated tissues were incubated in vitro in the presence of 14C-3-FMP. Studies with a range of metabolic inhibitors demonstrated that accumulation into olfactory tissue in vitro was virtually abolished by metyrapone and SKF-525A, indicating a key role of cytochrome P-450 mediated metabolism in this process. This was substantiated further by the in vivo inhibition of accumulation by metyrapone. Studies on the in vitro metabolism of 14C-3-FMP by isolated rat olfactory tissue demonstrated the major metabolite to be 3-trifluoromethylpyridine-N-oxide (3-FMP N-oxide) which is known to cause olfactory and hepatic toxicity in the rat. Metyrapone, while inhibiting accumulation of radioactivity derived from both 14C-3-FMP and 14C-3-FMP N-oxide in this tissue in vitro, only inhibited the synthesis of this metabolite by approximately 60%, indicating that several metabolic stages are involved in the metabolism and accumulation of 14C-3-FMP.

Methyl methacrylate toxicity in rat nasal epithelium: investigation of the time course of lesion development and recovery from short term vapour inhalation.

An investigation of the time course of development and recovery of the nasal lesion induced in rats by inhalation of methyl methacrylate (MMA) was conducted. Groups of 45 female F344 rats (five animals per time point) were exposed whole body for 6 hours per day to 0 (control), 110 or 400 ppm MMA for 1, 2, 5, 10 or 28 consecutive days. Additional animals were retained for a period of 4, 13, 24 or 36 weeks following exposure to assess reversibility of any nasal tissue effects. After inhalation of MMA there was damage to the olfactory epithelium at 110 and 400 ppm. This was apparent following the first day of exposure, but recovery/regeneration was evident during the subsequent days of the exposure phase of the study. The most severely affected section of the nasal passages was that which included the ethmoturbinates. Focal adhesions between the septum and turbinates and between the turbinates themselves were seen in some animals exposed to 400 ppm MMA at time points after 5 days of exposure. There were no lesions in the squamous, transitional or respiratory epithelia and none in control rats. Lesions that developed in rats exposed to 110 ppm MMA subsequently repaired during the exposure period. At 400 ppm, the majority of the olfactory epithelium had returned to normal within 13 weeks of the end of the exposure phase, but minimal respiratory metaplasia remained evident and there were some focal adhesions between the septum and turbinates and between the turbinates themselves.

Formic acid excretion in rats exposed to trichloroethylene: a possible explanation for renal toxicity in long-term studies.

Rats exposed to trichloroethylene, either by gavage or by inhalation, excreted large amounts of formic acid in urine which was accompanied by a change in urinary pH, increased excretion of ammonia, and slight increases in the excretion of calcium. Following a single 6-h exposure to 500 ppm trichloroethylene, the excretion of formic acid was comparable to that seen after a 500 mg/kg dose of formic acid itself, yet the half-life was markedly different. Formate excretion in trichloroethylene treated rats reached a maximum on day 2 and had a half-life of 4-5 days, whereas urinary excretion was complete within 24 h following a single dose of formic acid itself. Formic acid was shown not to be a metabolite of trichloroethylene. When rats were exposed to 250 or 500 ppm trichloroethylene, 6 h/day, for 28 days, the only significant effects were increased formic acid and ammonia excretion, and a change in urinary pH. There was no evidence of morphological liver or kidney damage. Long-term exposure to formic acid is known to cause kidney damage suggesting that excretion of this acid may contribute to the kidney damage seen in the long-term studies with trichloroethylene.

Olfactory and hepatic changes following inhalation of 3-trifluoromethyl pyridine in rats.

Rats exposed by inhalation to 3-trifluoromethylpyridine (3-FMP) for 10, 30 or 90 days showed an unusual response in the nasal passages. Focal histological change including reduction in the number of cell layers, disorganisation, vacuolation and minimal necrosis was confined to the olfactory epithelium. Axon bundles and the olfactory bulb were unaffected but there was loss of PAS staining affinity in Bowman's glands. The onset of the lesion showed a very steep dose-relationship approximating a quantal response; no effects were seen after 90 days exposure to 0.1 ppm but the changes were fully developed after 30 days exposure to 0.5 ppm. There was no marked progression with either increased exposure concentrations (up to 329 ppm) or with increased duration of exposure (10-90 days). The respiratory epithelium was generally unaffected apart from a mild irritant response seen only after 90 days. Exposures also resulted in a response in the liver. Centrilobular and midzonal vacuolation was observed at 10 and 30 days following exposures at or above 5 ppm 3-FMP and the severity increased with concentration. The lesion regressed with time even when exposure continued and only minimal changes were evident after 90 days, probably indicating an adaptive response. This work demonstrates the high organ specificity of 3-FMP, particularly for the olfactory epithelium.

Olfactory and hepatic changes following a single inhalation exposure of 3-trifluoromethyl pyridine in rats: concentration and temporal aspects.

The effects of a single exposure to 3-trifluoromethyl pyridine (3FMP), were investigated in two studies. In the first study, rats were exposed nose only to 0, 50 or 800 ppm 3FMP for periods of 15 min to 4 h. Half were sacrificed on day 3 and the remainder on day 10. In the second study, rats were exposed whole body to 0, 0.1, 1.0, 10 or 50 ppm 3FMP for 6 h, with sacrifices immediately after exposure (6 h), 24 h and on days 3 (48 h after exposure started) 5, 8, 11, 35, 70 and 157. Effects were seen in the olfactory epithelium at concentrations of 1 ppm and above and in the liver at concentrations of 50 ppm and above. In the olfactory epithelium the earliest changes were seen immediately after exposure and by 24 h this progressed to extensive necrosis with sloughing of the epithelium. By day 3, the epithelium was replaced by undifferentiated basophilic cells, considered to reflect early regeneration. Regeneration progressed to complete recovery between days 70 and 157, no changes were seen in the nasal respiratory epithelium, an olfactory function test on rats exposed for 6 h to 50 or 10 ppm 3FMP showed a reduced sense of olfaction at days 3 and 5 with complete recovery on subsequent days, indicating functional recovery in advance of histological normality. Single cell necrosis was seen in the liver at day 3 after 30 min exposure and immediately after 6 h exposure to 50 ppm 3FMP. At 24 h after a 6 h exposure to 50 ppm this had progressed to necrosis, haemorrhage and moderate cytoplasmic hepatocyte vacuolation in centrilobular areas. The lesion had completely recovered by day 5.

Induction of respiratory hypersensitivity to diphenylmethane-4,4'-diisocyanate (MDI) in guinea pigs. Influence of route of exposure.

The induction of respiratory sensitization in guinea pigs to diphenylmethane-4,4'-diisocyanate (MDI), a known human respiratory allergen, has been investigated and different routes of exposure compared. Guinea pigs were exposed to MDI by i.d. injection, by topical application or by inhalation. Pulmonary hypersensitivity was measured subsequently as a function of changes in respiratory rate following challenge with atmospheres containing MDI. In addition, contact hypersensitivity was measured by topical challenge and antibody responses evaluated by enzyme-linked immunosorbent assay (ELISA) and passive cutaneous anaphylaxis (PCA). Attempts to sensitize guinea pigs by inhalation exposure to MDI were unsuccessful. Antibody responses and contact sensitization were both infrequent and low grade, and no animals exhibited pulmonary responses following challenge with atmospheric MDI. In contrast, sensitization by either i.d. injection or topical application of MDI induced antibody responses in the majority of animals. Moreover, a proportion of animals in each case exhibited pulmonary responses following subsequent inhalation challenge. These data indicate that the route of exposure influences markedly the effectiveness of sensitization to respiratory allergens such as MDI and that skin contact may be an important cause of occupational respiratory allergy.

A two-centre study for the evaluation and validation of an animal model for the assessment of the potential of small molecular weight chemicals to cause respiratory allergy.

This study evaluated a single intradermal injection model in the guinea pig with subsequent inhalation challenge and serological analysis as a method to predict the potential of chemicals to induce respiratory allergy. Four known respiratory allergens (trimellitic anhydride, diphenyl methane diisocyanate, phthalic anhydride and toluene diisocyanate (TDI)) were screened by two industrial research laboratories using this protocol. Dinitrochlorobenzene, a potent contact allergen, was included as a negative control material. In both laboratories, the respiratory allergens, but not the contact allergen, induced high titre antigen-specific antibodies in treated animals. The inhalation challenge results were similar in both laboratories but were less conclusive in that exposure to free TDI failed to induce pulmonary responses, probably because it fails to penetrate to the deep lung in sufficient concentration. Although the assay shows promise as a means of identifying chemical respiratory sensitisers, its use as a routine screen for the prediction of the ability of materials to induce respiratory allergy in man is probably questionable.

Sensitisation of guinea pigs by inhalation exposure to low molecular weight chemicals.

Guinea pigs could be immunologically sensitised (as shown by the development of antigen-specific homocytotropic antibodies) to toluene diisocyanate by exposing them for 3 h a day for 5 consecutive days to atmospheres containing free chemical. Pulmonary reactions could be elicited in many of the sensitised animals by challenging them with atmospheres containing protein conjugates of the chemical and then measuring changes in respiratory rate. Successful elicitation of pulmonary reactions appeared to depend upon a number of factors, including the quality of the protein conjugate used for the challenge, but possibly also the development of IgE as well as IgG1 antibodies. Antigen-specific homocytotropic antibodies were detected in guinea pigs similarly exposed by inhalation to two non-isocyanate respiratory allergens, trimellitic anhydride and a reactive dye. Although the animals were immunologically sensitised to the chemicals, challenge with atmospheres containing appropriate chemical-protein conjugates failed to stimulate changes in respiratory rate.

The induction of respiratory allergy in guinea-pigs following intradermal injection of trimellitic anhydride: a comparison with the response to 2,4-dinitrochlorobenzene.

Guinea-pigs injected intradermally with the known respiratory sensitiser trimellitic anhydride (TMA) developed high-titre antigen-specific homocytotropic (IgG1 and IgE) antibodies. Many of the sensitised animals responded to a challenge by inhalation with either free TMA or a TMA-protein conjugate with a change in respiratory rate, reflecting the onset of bronchoconstriction. Guinea-pigs were also injected intradermally with 2,4-dinitrochlorobenzene (DNCB), which is a potent skin sensitiser in man but which has not been reported to cause respiratory allergy. These animals developed only low-titre homocytotropic antibodies and were unresponsive to an inhalation challenge with either free or conjugated hapten. The animals were, however, contact-sensitised to the chemical.

Current perspectives on particulate induced pulmonary tumours.

1. Chronic exposure to insoluble particulates can lead to the development of pulmonary tumours. These have been classified as broncho-alveolar or squamous/epidermoid according to their histopathological characteristics and have been reported in inhalation studies in rats of materials ranging from diesel exhaust and silica to titanium dioxide. 2. The sequence of changes within the rat lung leading to tumours has been characterised. It is apparent that one prerequisite is that the lung load of the particulate matter must exceed the normal clearance capacity, either by overloading the normal alveolar macrophage mediated mechanism or by induction of toxicity with materials such as silica. This results in inflammatory responses, including, or resulting in, epithelial hypertrophy and/or hyperplasia and squamous metaplasia. The persistence of these tissue responses over chronic time periods can lead to tumorigenesis. 3. Research into the mechanisms involved in the initiation and progression of both the inflammatory response and subsequent tumorigenic response to lung particulate loading is in progress. Impairment of macrophage function and mobility by inert particles constitutes one route by which this can arise, as does toxicity to this cell type by biologically reactive particles. At the molecular level, the role of inflammatory mediators, especially the cytokines, has received much attention. 4. Particulate induced lung tumours are perceived to be a phenomenon specific to the rat and their relevance to man is questionable.

Absorption, distribution, metabolism and excretion of an inhalation dose of [14C] 4,4'-methylenediphenyl diisocyanate in the male rat.

The received dose, tissue distribution, metabolism, routes and rates of excretion of [(14)C]-4, 4(')-methylenediphenyl diisocyanate (MDI) were investigated in the male rat following a 6-h inhalation exposure to [(14)C]-MDI at a target concentration of 2 mg m(-3). The mean dose received was equivalent to 0.078 mg MDI per animal, of this between 25 and 32% of radiolabelled material was available systemically. Radioactivity was distributed to all tissues examined with the highest proportions present in the respiratory and gastrointestinal tracts, suggesting that both oral ingestion and pulmonary absorption contributed to the systemic dose of [(14)C]-MDI derived material, with the oral ingestion and the majority of the internal dose resulting from ingestion of radiolabelled material by grooming the pelt after exposure. Radioactivity was excreted mainly via faeces (about 80% of the received dose). Excretion in bile and urine each accounted for less than 15% of the dose. MDI was extensively metabolized after uptake, with two routes of transformation evident; the proposed spontaneous formation of mixed molecular weight polyureas and the enzyme catalysed metabolism of systemically available MDI or MDI derivatives to give N-acetylated and N-acetylated hydroxylated products. No free MDA was detected in any of the biomatrices (urine, faeces, bile) investigated.

Biochemical effects of asbestiform minerals on lung fibroblast cultures.

The ability of different types of asbestiform minerals to enhance or suppress the levels of fibrous collagen in cultures of lung fibroblasts was tested. All commercially important asbestos dusts produced both effects, a lower dose favouring a fibrogenic response whereas a higher dose favoured an opposite effect.

The Sulphation of p-hydroxyphenylpyruvic acid and related compounds by the rat liver cytosol.

Cytosol preparations of rat liver and kidney were examined for their ability to transfer sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to p-hydroxyphenylpyruvic acid. Little activity towards this substrate was observed, and the main product detected in the reaction mixtures was identified as p-hydroxybenzyl alcohol O-sulphate. This was not formed from p-hydroxybenzaldehyde, a spontaneous oxidation product of p-hydroxyphenylpyruvic acid, by sulphation followed by a rapid enzyme-catalysed reduction of the intermediate phydroxybenzaldehyde O-sulphate. This product was formed mainly by this sequence of reactions, but the reverse, reduction followed by sulphation, also appeared possible. p-Hydroxybenzyl alcohol itself was very readily sulphated by both preparations, and the liver also produced a sulpho-conjugate of homogentisic acid. These observations are important in calculating the turnover of L-tyrosine O-sulphate in the mammalian system, and establish that p-hydroxyphenylpyruvic acid O-sulphate is an end product of its metabolism, rather than an intermediate in its synthesis by reversed transamination.

Subchronic feeding study of antimony trioxide in rats.

Diets containing antimony trioxide were fed to male and female Wistar rats of the Alpk:APSD strain over 90 days. Dose levels were 0 (control), 1000, 5000 and 20,000 ppm (equivalent to mean daily doses of 84, 421 and 1686 mg kg(-1) in males and 97, 494 and 1879 mg kg(-1) in females). There was no effect of compound on growth or growth rate, food consumption or clinical signs. Minor changes in haematology and urine biochemistry were considered incidental to treatment. Small reductions in plasma alkaline phosphatase activity and increases in aspartate aminotransferase activity at the high dose, together with a small (ca. 10%) increase in liver weight, could be indicative of a minor effect on the liver, but in the absence of any histological effects these changes are also considered incidental to treatment. This study confirms the inert nature of antimony trioxide.

The role of trichloracetic acid and peroxisome proliferation in the differences in carcinogenicity of perchloroethylene in the mouse and rat.

Fischer 344 rats and B6C3F1 mice of both sexes were exposed to 400 ppm perchloroethylene (PER) by inhalation, 6 hr/day for 14, 21, or 28 days or to 200 ppm for 28 days. Increased numbers of peroxisomes were seen under the electron microscope and increased peroxisomal cyanide-insensitive palmitoyl CoA oxidation was measured (3.6-fold increase in males and 2.1-fold increase in females) in the livers of mice exposed to PER. Hepatic catalase was not increased. Peroxisome proliferation was not observed in rat liver or in the kidneys of either species. Trichloracetic acid (TCA), a known carcinogen and hepatic peroxisome proliferating agent, was found to be a major metabolite of PER. Blood levels of this metabolite measured in mice and rats during and for 48 hr after a single 6-hr exposure to 400 ppm PER showed that peak blood levels in mice were 13 times higher than those seen in rats. Comparison of areas under the curves over the time course of the experiment showed that mice were exposed to 6.7 times more TCA than rats. The difference in metabolism of PER to TCA in mice and rats leads to the species difference in hepatic peroxisome proliferation which is believed to be the basis of the species difference in hepatocarcinogenicity. Peroxisome proliferation does not appear to play a role in the apparent carcinogenicity of PER in the rat kidney.

Determination and significance of l-tyrosine O-sulphate and its deaminated metabolites in normal human and mouse urine.

Methods were developed for the determination of the O-sulphate esters of l-tyrosine, p-hydroxyphenylpyruvate, p-hydroxyphenylacetate and p-hydroxybenzaldehyde in human urine. The amounts of these esters normally present in human female urine were determined. The quantities and specific radioactivities of l-tyrosine O-sulphate and p-hydroxyphenylpyruvate O-sulphate in mouse urine after labelled tyrosine had been fed were determined and were consistent with the hypothesis that the sole source of p-hydroxyphenylpyruvate O-sulphate was l-tyrosine O-sulphate. It is suggested that the turnover of known polypeptides containing l-tyrosine O-sulphate residues can account for only a portion of the quantities of the ester and its metabolite(s) that are present in normal female human urine.

1,1,1,2-Tetrafluoroethane: repeat exposure inhalation toxicity in the rat, developmental toxicity in the rabbit, and genotoxicity in vitro and in vivo.

Subchronic and chronic studies were carried out in the rat and a developmental toxicity study in the rabbit with exposures to 1,1,1,2-tetrafluoroethane (HFC 134a) by inhalation. In the rat repeated exposure to 50,000 ppm HFC 134a for 13, 52, and 104 weeks elicited no effect on clinical condition, growth, and survival, or on a variety of hematological, clinical chemistry, and urinary parameters. Treatment-related pathological changes were seen only at study termination at 2 years and were confined to increased incidence of Leydig cell hyperplasia and adenoma in male rats exposed to 50,000 ppm. The tumors, which were also seen in control animals, were benign and not life-threatening. A battery of in vitro and in vivo tests gave no evidence of genotoxic activity. With exposure to pregnant rabbits, the only treatment-related effects were of minimal maternal toxicity at high exposure concentrations; there were no effects on fetal development. It is concluded that HFC 134a is of very low toxicity and should be an acceptable alternative to CFCs.

Characterization of pulmonary surfactant from ox, rabbit, rat and sheep.

1. Pulmonary surfactants from ox, rabbit, rat and sheep were isolated and analysed. 2. All preparations had a high anenoic phosphatidylcholine content and would produce stable surface tensions of 0.01 Nm-1 or less. 3. Protein content was 8-18% of the dry weights. A number of proteins were observed; their overall composition were high in hydrophobic amino acid residues. 4. Lipid content varied from 79% (ox) to 90% (rabbit) with phosphatidylcholine representing from 58% (sheep) to 83% (rabbit) of the total lipid. The surfactant preparations were rather similar in lipid composition except that sheep surfactant contained about 10% lysophosphatidylcholine. 5. Hexadecanoic acid was the principal fatty acid. It was particularly high in phosphatidylcholine. 6. Phosphatidylglycerol was a minor constituent of all surfactants but phosphatidyldimethylethanolamine was not detected.

Chrysotile-induced asbestosis: changes in the free cell population, pulmonary surfactant and whole lung tissue of rats.

Rats inhaling chrysotile asbestos contracted asbestosis and fibrosis of the lungs. Studies of biochemical and morphological changes (between normal and treated animals) show that chrysotile induces an increase in the lung free cell population and pulmonary surfactant levels. Lysosomal enzyme levels are elevated in both the whole lung and free cell population and there are considerable changes in macrophage morphology. It is suggested that the primary response of the lung to chrysotile is an increase in surfactant production coupled with an increase in free cell numbers, in order to prevent the cytotoxic effect of the dust.