Priority Existing Chemical

j.8Pulmonary toxicity

Hanzlik et al. (1980a) investigated the pulmonary toxicity of MMT in male Sprague-Dawley rats. Single oral dosing with 125 mg/kg bw MMT (in corn oil) resulted in haemorrhage and alveolar and perivascular oedema of the lung after 24 hours. An accumulation of proteinaceous material in the alveoli characterised the alveolar oedema. Lung weight, a measure of pulmonary toxicity, was significantly elevated 24 and 72 hours after oral dosing of MMT (125 mg/kg bw) and 8/14 rats died within 24 hours. The lungs of the deceased rats showed extensive haemorrhaging and congestion. There were minor lesions in the liver and kidneys. Lung weight was also significantly elevated 24, 48, and 72 hours after IP injection of MMT (20 mg/kg bw) in corn oil. Pre-treatment with phenobarbital (60 mg/kg bw for 3 days) protected against the pulmonary toxicity of MMT but induced mildly increased plasma GPT and decreased liver G6P indicating liver damage. The authors postulate that the protective effect of phenobarbital may be due to a first-pass effect where an enlarged, metabolically-induced liver limits the amount of MMT entering the systemic circulation. Between 3 and 14 animals were used per treatment.

The effect of MMT on Clara cells was examined after a single IP injection to female BALB/C mice (120 mg/kg bw), female S/A albino rats (5 mg/kg bw) and female LV₆/LAK Syrian hamsters (180 mg/kg bw). The incorporation of radiolabelled thymidine into pulmonary DNA, as a measure of cellular proliferation, was found to decrease slightly one day post MMT administration in mice and hamsters. All species showed a significant increase in thymidine incorporation by day 2, with peak incorporation at day 2 in rats and hamsters and day 4 in mice. Labelling indices in rats and mice, as determined by cell kinetic studies, demonstrated a significant increase in bronchiolar and parenchymal indices at day 2. Interstitial pneumonitis characterised by interstitial thickening and infiltration by neutrophils and macrophages, was observed in all animals two days post MMT injection but all were found to clear by day 21. With respect to interstitial pneumonitis, rats were the most sensitive species to MMT treatment, followed by mice, and then hamsters. Clara cell necrosis was evident in all species one day post MMT administration. This effect was greatest in distal airways and was most severe in mice, which had failed to return to normal by day 21. Bronchiolar morphology had returned to normal by day 7 in the hamster and day 21 in the rat. In addition, necrosis of the tubular epithelium in the renal cortex was observed in mice and minimally in the other species on days 1 and 2 post MMT administration. The number of animals per treatment totalled 36 for mice and 18 for rats and hamsters (Hakkinen and Haschek 1982).

The effect of MMT on Clara cells was also examined after a single IP injection to female BALB/C mice (120 mg/kg bw) and male Fischer-derived rats (8.4 mg/kg bw). Histopathological analysis 24 hours post MMT injection revealed a selective necrosis of Clara cells, particularly at terminal bronchioles. Mice were found to be more sensitive than rats. The pulmonary toxicity of MMT (90 mg/kg bw) was also assessed in female mice after a 1 hour pre-treatment with piperonyl butoxide, an inhibitor of the mixed-function oxidase system. The pre-treatment was found to significantly enhance the toxicity of MMT. Clara cell damage extended into the larger bronchioles and moderate oedema, inflammation and localised haemorrhaging became apparent within the parenchyma. In addition, the incidence of mortality was increased. The number of animals used per treatment was not stated (Haschek et al., 1982).

The ability of oxygen to enhance the pneumotoxic effects of MMT was investigated by Hakkinen et al. (1983) in female BALB/C mice and female CD/CR rats. Animals were injected IP with MMT in corn oil (5 mg/kg bw for rats and 120 mg/kg bw for mice) and immediately dosed with either 80% oxygen or air for 6 days. Total lung hydroxyproline levels were unaffected by MMT in mice 3 weeks after MMT dosing. A significant increase in total lung hydroxyproline was observed in treated mice when MMT was combined with oxygen treatment. The lungs of these mice were found to contain scattered areas of interstitial thickening characterised by a mild hypercellularity and fibrosis, located mainly at terminal bronchioles and alveolar ducts. Although MMT was found to significantly increase total lung hydroxyproline levels in rats 3 weeks post MMT dosing, this effect was not enhanced by oxygen. Fibrosis noted in the lungs of rats exposed to both MMT and oxygen was similar in rats treated with only MMT. Between 8-10 mice and 4-6 rats were used per treatment (Hakkinen et al., 1983).

The toxicity of MMT was examined in male CD rats 1.5-96 hours after subcutaneous administration of a single dose of MMT (4 mg/kg bw in propylene glycol). Pulmonary lavage protein levels were used as an indicator of pulmonary toxicity. Pulmonary lavage protein levels were significantly elevated at all time points examined with levels peaking (5-fold increase) indicating maximal pulmonary toxicity at 24-48 hours after MMT administration. Pulmonary Mn levels peaked 3-6 hours post injection. Plasma urea and sorbitol dehydrogenase levels were not significantly altered by MMT, indicating little or no hepatic or renal injury. The number of animals used per treatment was not stated (McGinley et al., 1987).

Cox et al. (1987) examined the pulmonary toxicity of MMT in male Sprague-Dawley rats. A single IP injection of MMT (6-37.4 mg/kg bw) resulted in extensive mottling, haemorrhage and distension of the lungs and the presence of a pink, frothy, serosanguineous liquid in the trachea. Four animals were used per treatment.

A single subcutaneous injection of MMT (4 or 10 mg/kg bw) in corn oil vehicle to male Sprague-Dawley rats resulted in significant pneumotoxic responses. No deaths were observed at the lower dose. At the highest dose, 1/6 of the rats died within 24 hours. Laboured breathing prior to death and the presence of a frothy fluid in the trachea at necropsy were observed. Hepatic and renal markers (plasma lactate dehydrogenase, sorbitol dehydrogenase, and blood urea nitrogen) were considered normal. The lavage fluid of rats surviving for 24 hours after MMT administration (10 mg/kg bw) contained increased lactate dehydrogenase, albumin and total protein. These results correlate well with increased lung Mn content resulting from MMT administration (See Section 9). An additional experiment was performed assessing pneumotoxic responses resulting from MMT (4 mg/kg bw) administration in the presence of piperonyl butoxide, a cytochrome P450 monooxygenase inhibitor. A 1-hour pre-treatment with piperonyl butoxide (400 mg/kg bw) was found to protect against increases in lavage albumin and reduce the levels of Mn in the lungs. Pulmonary nonprotein sulfhydryl levels were significantly increased 24 hours after administration of MMT at 4 mg/kg bw, but this effect was reduced by piperonyl butoxide. The level of pulmonary thiobarbituric acid reactive materials was not altered by MMT. Heptane extraction of lung homogenates from MMT-treated rats indicated less than 2% of the pulmonary Mn was extractable, suggesting the presence of metabolites as opposed to MMT. Furthermore, the decrease in pneumotoxicity in the presence of piperonyl butoxide suggests that cytochrome P450 dependent monoxygenase metabolites are responsible for toxicity. Each treatment group contained 4-9 animals (Clay and Morris 1989).

The pneumotoxicity of MMT in female LAC-P Wistar rats was investigated after a single IP injection (6 mg/kg bw in oil). Lung weight and the activity of -glutamyltranspeptidase and alkaline phosphatase in bronchoalveolar lavage fluid were quantified as measures of pulmonary toxicity. The lung wet weight of MMT-treated rats was approximately twice that of the controls at 3-5 days post administration. MMT also resulted in a significant increase in the activity of the bronchoalveolar lavage fluid enzyme alkaline phosphatase 24 hours post administration. Pneumotoxic effects consisted of a loss of type I pneumocytes, proliferation of type II cells and macrophage infiltration. Inhibitors of the cytochrome P-450 2B isoenzyme (O,O,S-trimethylphosphorodithioate, bromophos, 2,4-dichloro(6-phenylphenoxy)ethylamine and p-xylene) protected against the pulmonary toxicity of MMT. Five animals were used per treatment (Verschoyle et al., 1993).

The effect of MMT on Clara cells of the small airways was investigated in Sprague-Dawley rats. Each experimental group consisted of six rats. In this study rats were sacrificed 24 hours after a single IP injection of MMT (5 mg/kg bw). The lungs were lavaged and bronchoalveolar lavage fluid (BALF) was isolated and analysed for biochemical markers. MMT was found to cause a significant decrease in 16-17 kDa Clara cell protein (CC16) in BALF and a significant increase in albumin content. MMT did not alter the serum concentration of CC16 or renal function parameters (serum creatinine and albuminuria). However, the concentration of CC16 in urine was significantly increased. Histopathological analysis of the lung parenchyma revealed a slight interstital thickening and mild oedema in the alveolar walls and perivascular connective tissue. An increase in the numbers of enlarged type II pneumocytes and alveolar macrophages were observed in the alveolar lining and alveolar spaces respectively. The number and activity of neutrophils and lymphocytes appeared normal. Although ciliated cells of the epithelial lining of bronchioles appeared normal, Clara cell necrosis was evident, especially in distal airways (Halatek et al., 1998).