Shigeo Kashiwamata

Dangerous effects of tin-protoporphyrin plus photoirradiation on neonatal rats

In vivo and in vitro effects of porphyrins (tin-protoporphyrin [SnPP], cobalt-mesoporphyrin, haemin and protoporphyrin) on neonatal rats were investigated. Under photoirradiation a high mortality rate was recognized in SnPP injected rats. None died from the application of SnPP without photoirradiation. In photoirradiated rats the median lethal dose (LD50) value of SnPP was calculated to be about 7.4 μmol/kg body weight. Haemolysis and malonaldehyde formation of red blood cells were induced by SnPP together with photoirradiation. SnPP may be useful in reducing bilirubin levels in severely jaundiced infants under non-photoirradiated conditions or dim light, but prophylactic administration of SnPP to the majority of infants is not recommended.

Two proteins associated with cerebellar hypoplasia in jaundiced Gunn rats

Two cerebellar proteins with apparent molecular weights of 250,000 (GR-250) and 50,000 (GR-50) are closely associated with cerebellar hypoplasia in jaundiced homozygous Gunn rats. These proteins, found in Gunn rat cerebellum (4–60 days of age) and cerebrum as well as staggerer mouse cerebellum, were studied with electrophoretic techniques. After 8 days of life, GR-250 decreased and GR-50 increased in the homozygous Gunn rat cerebellum. The pIs of GR-250 and GT-50 were 4.7–5.8 and 4.6–4.9, respectively, and the former protein was shown to bind to Concanavalin A. A comparative study between cerebella of Gunn rats and staggerer mice revealed that GR-250 and P400, a protein generally thought to be characteristic of the Purkije cells, were identical. Evidence was also obtained showing that GR-250 was present in the Gunn rat cerebrum. GR-50 was not detectable in the staggerer mouse cerebellum but instead, a protein (MW 47,000) was found to be increased in the mutant mouse cerebellum.

Effect of Irradiation on the Erythrocyte Membranes of Homozygous Gunn Rat Sucklings

Erythrocyte membranes were isolated from irradiated Gunns strain of jaundiced rat sucklings, and photodynamic action of bilirubin in vivo was investigated using sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The effectiveness of irradiation was verified in terms of decrease of plasma bilirubin concentrations and cerebellar bilirubin contents, and also by recovery from the cellbellar hypoplasia, a characteristic feature of the homozygotes (j/j). The cross-linking of erythrocyte membrane proteins by photodynamic action of bilirubin was demonstrated in vitro in rat erythrocytes as reported for human erythrocytes by Girotti (Biochemistry 14: 3377, 1975). The phenomenon, however, was not observed in vivo. Instead, a protein band with a mol. wt. of 32,000 was increased by the irradiation both in the homozygous (j/j) and heterozygous (j/+) Gunn rats.

Protection by L-ascorbic acid against phototoxicity in tin-protoporphyrin-treated suckling rats

The protective effect of free radical scavengers against phototoxicity was investigated with tin-protoporphyrin (SnPP)-treated suckling rats. Six kinds of scavengers (L-ascorbic acid, reduced glutathione, alpha-tocopherol, retinol, uric acid and cystine) were intraperitoneally injected to rats treated with SnPP plus photoirradiation. Among them, L-ascorbic acid was found to be most effective in protecting SnPP-treated rats against phototoxicity. The survival period was markedly prolonged, and the frequency of abnormal behaviors was reduced with the treatment. Lipid peroxidation in vitro with the brain membrane fraction was also suppressed. The other five substances gave only a little antioxidant effect both in vivo and in vitro. The present study shows that L-ascorbic acid may be a promising chemical to prevent the phototoxicity of SnPP.

A low-molecular-weight cadmium-binding substance in human and rat livers and human blood

A cadmium-binding substance with a molecular weight even lower than that of metallothionein was demonstrated on Sephadex G-75 gel filtration chromatography of the soluble fractions from newborn human and adult rat liver homogenates and adult human hemolysate which were mixed with CdCl2 in vitro. This substance was purified from rat liver extracts by gel filtration and ion-exchange column chromatography and characterized by 6 M guanidine X HCl thin-layer gel filtration chromatography and N-terminal and total amino acid analyses. The results showed that the isolated low-molecular-weight cadmium-binding substance was a cadmium-reduced glutathione complex, whose molecular weight was found to be approximately 1400 by Sephadex G-15 gel filtration.

An acceleration of age-related increases in levels of the β-subunit of nerve growth factor in selected tissues from senescence-accelerated mice (SAM-P/8)

An investigation was made of age-related changes in levels of the β-subunit of nerve growth factor (β-NGF) in selected tissues and of testosterone in serum in senescence-accelerated mice (SAM-P/8) and in the control mice (senesence-resistant mice; SAM-R/1). The concentrations of testosterone in serum were higher in SAM-P/8 than in SAM-R/1 at ages 2 and 4 mo. The level of β-NGF in the thymus from SAM-R/1 increased with age, resulting in a statistically significant difference in its level between mice at ages 2 and 12 mo. By contrast, there was a transient increase in SAM-P/8 at around age 4 mo with a subsequent decrease. Consequently, significant differences were apparent in levels of β-NGF between the two types of mouse at ages 2 and 4 mo. Similar results were obtained in the adrenal gland and testis. Compared to SAM-R/1 at age 2 mo, the average concentrations of β-NGF in the hypophysis were higher in SAM-R/1 at ages 4 and 8 mo and in SAM-P/8 at all ages. In other tissues tested, no remarkable differences were detected. Our present results indicate that, in SAM-P/8, the elevation in levels of β-NGF in the thymus, adrenal gland, testis, and hypophysis occurs in the early period of life compared to the control mice. Possible dysfunction of the disorder of hypophysis is discussed.

Pharmacological and Biological Effects of Tin-Protoporphyrin on Neonatal Hyperbilirubinemic Gunn Rats

ABSTRACT: Our study was undertaken to examine the pharmacological and biological effects of tin-protopor-phyrin, a competitive inhibitor of heme oxygenase, on 5-or 6-day-old homozygous (j/j) Gunn rats with hereditary unconjugated hyperbilirubinemia. When j/j neonates were injected subcutaneously with 20 μmol of tin-protopor-phyrin/kg of body weight, hepatic heme oxygenase activity decreased to 30% of the initial level 2 h after administration and remained low during the next 46 h. However, the reduction of serum bilirubin was more rapid and transient, reaching the minimum value (40% of the initial level) at 1 h and increasing thereafter at a rate almost comparable to that in nontreated j/j rats. The mortality rate of j/j rats was strikingly reduced by the administration of 1 to 100 μmol of tin-protoporphyrin/kg; the most effective dose was 5 μmol/kg (8% compared with 80% in non-treated j/j rats). However, the protective effect of tin-protoporphyrin on bilirubin cerebellopathy (cerebellar hypoplasia) was less marked than expected. Possible implications of our results are discussed.

Cobalt-mesoporphyrin inhibits heme oxygenase activity but it does not induce lipid peroxidation in rat brain membranes during photoirradiation.

We examined the effects of cobalt-mesoporphyrin (CoMP) in vitro. The porphyrin inhibited the activity of rat splenic heme oxygenase but scarcely stimulated peroxidation of lipids in a membrane fraction from rat brain during photoirradiation. The apparent inhibition constant for CoMP was 344 nM. It is suggested that CoMP may be a promising candidate for a chemopreventive of neonatal hyperbilirubinemia that is not associated with phototoxicity.

Mode of Bilirubin Deposition in the Cerebellum of Developing Jaundiced Gunn Rats

Bucolome is known as a potent displacer of bilirubin from the blood into tissues. The effects of the drug on newborn rats with congenital jaundice (Gunn rats) were examined on postnatal days 7, 11, 15 and 21. A single subcutaneous injection of bucolome resulted in a rapid fall of the total plasma bilirubin concentration and the lowered level persisted as long as 23 h. Concomitantly with the drop, the cerebellar bilirubin level increased within 1 h. Although cerebellar bilirubin returned to the initial level after 24 h in rats treated with bucolome on days 7 and 21, it remained almost constant or rather increased during the period of 1-24 h in rats treated on days 11 or 15. In 15-day-old rats, localized yellow staining in the cerebellum 24 h after treatment was most apparent in the granule cell layer of the ventral part of the pyramis and the dorsal part of the uvula under the dissecting microscope. These observations suggest that the period most susceptible to bilirubin deposition lies around day 15 in the cerebellum of jaundiced Gunn rats.

Thyroid hormone metabolism and nuclear binding in Gunn rats

We examined the serum concentrations of total, free thyroid hormones and TSH, activity of hepatic T45′-deiodinase, and T3 binding to hepatic nuclei in homozygous (j/j) and heterozygous (j/+) Gunn rats. Both total T3 and free T3 (FT3) concentrations in sera from j/j rats were significantly lower than those of j/+ rats on 5–10, 15–20, and 25–30 days after birth. Both total T4 and free T4 (FT4) concentrations in j/j and j/+ rat sera were not significantly different on 5–10 days. However, in j/j rats they were significantly higher than those of j/+ rats on days 15–20 and 25–30. Serum reverse T3 (rT3) concentrations were higher in j/j than in j/+ rats on days 5–10, 15–20, and 25–30. Serum TSH concentration in j/j and j/+ rats on 15 days post-natal were 1.42±1.28 and 1.65±1.24 µg/l (mean±SD), respectively, wich were not significantly different from each other. T3 formation from T4 in hepatic microsomal fractions obtained 15 days after birth was significantly lower in homozygotes than in heterozygotes (4.89±1.18 vs 11.15±2.38 pmol/mg protein/min, p < 0.005). Binding constants (Ka) as well as maximal binding capacities (MBC) for T3 of hepatic nuclei from 15 day-old j/j and j/+ rats were similar (ka; 3.58×109vs3.15×109 M−1, MBC; 0.316 vs0.380 pmol/mg DNA). From these results we suggest that decreased conversion from T4 to T3 is one of the major reasons for high serum levels of T4 and rT3, and low levels of T3 in j/j rats, and that nuclear T3 binding and pituitary TSH secretion are unaltered in j/j rats.