Kentaro Kubota

Studies on biochemical effects of nitrogen dioxide. II. Changes of the protective systems in rat lungs and of lipid peroxidation by acute exposure

This work was done to clarify the relation between the change of lipid peroxidation and the protective systems in lungs after NO2 exposures. JCL:Wistar 8-wk-old male rats were exposed continuously to 10 ppm NO2 for 2 wk. Lipid peroxidation, measured by ethane exhalation in the breath of the rats and by the reaction of thiobarbituric acid with lung homogenates, increased to a maximum at 3 d after a decline at 1 d, and then returned to the initial level (of d 0). Activities of glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), disulfide reductase (DSR), and superoxide dismutase (SOD) in the 105,000 X g supernatant of lung homogenates were depressed slightly at 1 d. Thereafter, they increased significantly to their maximum levels from 5 to 10 d, and these maximum levels were maintained until d 14. The pattern of change of these protective enzymes was symmetric to that of lipid peroxidation after 3 d. The order of the ratio of the increased value to the initial value was G6PD greater than DSR greater than 6PGD greater than GR greater than GPx greater than SOD. The time course of nonprotein sulfhydryls was similar to that of the protective enzymes. In contrast, the amounts of vitamin E increased to a maximum at 2 d and then returned to the initial level. The periodic change of vitamin E was similar to that of lipid peroxidation rather than that of the protective enzymes. These results suggest that the ability of the enzyme systems in lungs to protect against NO2 fluctuated in a complex manner and the activities of the protective enzymes varied inversely with lipid peroxidation.

Studies on the biochemical effects of nitrogen dioxide. IV. Relation between the change of lipid peroxidation and the antioxidative protective system in rat lungs upon life span exposure to low levels of NO2.

This study examined the relation between lipid peroxidation and the antioxidative protective system in lungs of rats exposed to low levels of nitrogen dioxide (NO2). JCL:male Wistar rats were exposed to 0, 0.04, 0.4, and 4 ppm NO2 for 9, 18, and 27 months. Lipid peroxidation measured by TBA method, increased significantly in the 4 ppm NO2 group of the 9-month exposure and in the 0.4 and 4 ppm NO2 groups of the 18-month exposure. The activity of glutathione peroxidase measured with hydrogen peroxide as substrate decreased significantly in the 4 ppm NO2 group of the 9-month exposure and in the 0.4 and 4 ppm NO2 groups of the 18-month exposure. Furthermore, the activities of two glutathione S-transferases, aryl and aralkyl S-transferase, also decreased in the 0.4 and 4 ppm NO2 groups of the 18-month exposure, but not in any groups of the 9-month exposure. The activity of glutathione peroxidase measured with cumene hydroperoxide as substrate did not show any significant changes in any NO2 group. The activities of glucose-6-phosphate dehydrogenase and glutathione reductase were significantly higher than those in the control group for the 9-month exposure. In the 18-month exposure, however, they showed a tendency to return to control level. The activities of superoxide dismutase and disulfide reductase upon NO2 exposure for 9 and 18 months were not different from control values. To confirm that lipid peroxidation was increased with greater NO2 concentrations and exposure times, ethane and pentane exhalation in breath as an index of lipid peroxidation was examined. Ethane exhalation increased significantly following 0.04, 0.4, and 4 ppm NO2 exposure for 9 and 18 months. Furthermore, ethane formation of rats exposed to 0.04 and 0.4 ppm NO2 for 27 months also increased to twice the control level. On the other hand, after exposure to 4 ppm NO2 for 27 months, ethane levels returned to control level. Pentane formation increased significantly only in the 0.04 and 0.4 ppm groups in the 18-month exposure. Ethane exhalation in rats exposed to 0.04, 0.12, and 0.4 ppm NO2 for 9 and 18 months was similar. These results suggested that the antioxidative protective ability was decreased with prolonged exposure, while formation of cytotoxic lipid peroxides was increased.(ABSTRACT TRUNCATED AT 400 WORDS)

Tolerance to cadmium and cadmium-binding proteins induced in the midge larva, Chironomus yoshimatsui (diptera, chironomidae)

Abstract 1. The midge larva ( Chironomus yoshimatsui ) was exposed to cadmium (10 μg Cd/ml) for 2 days. 2. A large portion of cadmium taken up rapidly into the insects was bound to the high molecular weight proteins and was rapidly discharged in control water. 3. Low molecular weight cadmium-binding protein was slowly induced in the larva by cadmium exposure. This protein was a mixture of four isoproteins and showed the characteristic properties of metallothionein. 4. The high tolerance of the midge larva to acute cadmium exposure was not explainable by induction of the cadmium-binding proteins.

Fate and comparative toxicity of metallothioneins with differing Cd/Zn ratios in rat kidney

Metallothioneins with differing Cd/Zn ratios were preparedin vitro from rat liver zinc-thionein by replacing zinc with cadmium and were injected intraperitoneally to female rats. The distribution of cadmium, zinc, and copper in the kidney supernatant fraction was determined using a Sephadex G-75 column. The distribution pattern of cadmium and zinc changed dramatically within 24 hr after the injection. The changes were explained by the degradation and re-synthesis of metallothionein in the kidneys. The necrotic changes of renal tubular lining cells were correlated to the amount of cadmium in the metallothionein but not to the amount of metallothionein (protein).

In vitro evaluation of cadmium-induced augmentation of the antibody response

Augmentation of in vitro primary plaque-forming cell (PFC) response by cadmium exposure was investigated. Spleen cells from 2- to 3-months-old BALBc mice were incubated with various concentrations of cadmium chloride in the presence of sheep red blood cells (SRBC) as an antigen. PFC responses were enhanced by 4 and 8 μm cadmium, but suppression of PFC responses was observed at concentrations of 20 and 40 μm cadmium. In the reconstitution of adherent and nonadherent cells to culture dishes from spleen cells which were incubated for 24 hr with 8 μm cadmium or saline (control), it was found that nonadherent cells were significantly enhanced by cadmium. Moreover, in the reconstitution between T and B cells from control and exposed groups, B cells from exposed groups were more enhanced by cadmium than T cells. These results suggested that augmentation of primary PFC response by cadmium exposure was mainly caused by the enhancement of B cells.

Studies on biochemical effects of nitrogen dioxide: I. Lipid peroxidation as measured by ethane exhalation of rats exposed to nitrogen dioxide

This research was in order to follow the periodic fluctuation of lipid peroxidation by a new method in rats exposed to nitrogen dioxide. Wistar male rats were examined for lipid peroxidation as demonstrated by ethane exhalation. In rats continuously exposed to 10 ppm nitrogen dioxide for 2 weeks, the amount of ethane exhaled fluctuated in a complex manner during the exposure. Ethane exhalation decreased slightly after the first day of exposure and then increased rapidly. The maximal values were observed after the fourth day of exposure and then decreased gradually to the initial level. Furthermore, the activity of glutathione peroxidase in lungs of rats exposed to 10 ppm nitrogen dioxide varied symmetrically against the change of ethane formation. Similar changes in ethane exhalation were observed in rats exposed to the lower levels of nitrogen dioxide (0.4, 1.2 and 4.0 ppm) for 4 months. Compared to 10 ppm nitrogen dioxide exposure for 14 days, the characteristics in rats exposed to the low levels (0.4–4.0 ppm) of nitrogen dioxide were: the decline of ethane formation, the delay in alterations, and the tendency toward gradual increased during the longer period exposure.

Alterations of nitrite and nitrate concentrations in the blood of mice exposed to nitrogen dioxide

Abstract Blood nitrite and nitrate of mice were determined using naphthylethylenediamine and a Cu-Cd reduction column. When mice were exposed to 40 ppm nitrogen dioxide, nitrite became constant in 10 min. Nitrite declined rapidly, with a half-life of several minutes, when mice were removed to room air. Nitrate showed changes similar to those of nitrite; however, the concentration in the blood was higher and the half-life was longer. Dose-effect relationships were also determined at concentrations ranging between 5 and 40 ppm for 1 hr exposure. No increase of methemoglobin was observed at these concentrations. Addition of fresh mouse blood to sodium nitrite in vitro indicated a rapid conversion of nitrite to nitrate with an increase of methemoglobin, whereas addition to sodium nitrate did not cause any changes. The fate of inhaled nitrogen dioxide in the living body is discussed based on the results obtained.

Accumulation of cadmium and induction of its binding protein in the digestive tract of fleshfly (Sarcophaga peregrina) larvae

The larva of Sarcophaga peregrina ( fleshfly ) was fed with cadmium (Cd)-containing diet and the distribution of Cd among tissues was determined by separating each organ. Approximately 90% of Cd accumulated in the larva was found in the digestive tract, the fat body and the Malpighian tube being less effective tissues in its accumulation. Cd in the digestive tract was mostly bound to an inducible Cd-binding protein. The Cd-binding protein was a mixture of five isoproteins having several properties characteristic of metallothionein.

Thymic atrophy in mice induced by cadmium administration

The effects of cadmium (Cd) on the immune organs were examined histopathologically. On 2 or 3 days after a single i.p. injection of 1.8 mg Cd/kg body weight into mice, slight loss of body weight, significant decrease of thymus weight and marked increase of spleen weight were observed. Lymph node weight did not show any change. Histopathologically, cortical atrophy of the thymus was very marked. The white pulp of the spleen tended to diminish in size any many polymorphonuclear leukocytes and myeloid cells appeared in the red pulp.

Effect of subacute exposure to NO2 on lymphocytes required for antibody responses

BALB/c mice were continuously exposed to 0.4 and 1.6 ppm NO/sub 2/ for 4 weeks and the effects on lymphocytes which are required for primary and secondary antibody responses to sheep red blood cells were examined in vitro. The primary antibody response was significantly suppressed by both concentrations of NO/sub 2/, whereas the secondary antibody response was slightly stimulated by 1.6 ppm NO/sub 2/ exposure. In reconstitution experiments no significant differences were observed in the activities of T and B lymphocytes from mice exposed to 1.6 ppm NO/sub 2/.