Hideaki Kito

Separation and purification of (Cd, Cu, Zn)-metallothionein in carp hepato-pancreas

1. Cd-binding protein was isolated from the hepato-pancreas of carp administered CdCl2 (2 mg/kg). 2. This protein had a high absorption at 254 nm derived from Cd-mercaptide binding, and a low absorption at 280 nm derived from the absence of aromatic amino acids; the authors recognized the presence of two types. 3. The amino acid composition of the proteins was determined. The contents of cysteine residues were 34.24% and 31.90% in MT-I and -II respectively. Tyrosine, phenylalanine, tryptophan, histidine, leucine and arginine residues were absent in both types.

Protection by metallothionein against cadmium toxicity

1. The protective effect against Cd toxicity of prior exposure to Cd or Zn solutions at low concentration was studied. 2. Carp were bred in tap water (A), 1 ppm Cd solution (B) and 5 ppm Zn solution (C) for 14 days and then transferred into 15 ppm Cd solution. The survival ratio of carp decreased in the order: (C):(B):(A). 3. Binding capacity of Cd to high molecular and metallothionein fractions in the cytoplasmic solutions of the hepato-pancreas was studied and the binding capacity to the metallothionein fraction was stronger than that to the high molecular fraction. The authors recognized that Zn in the metallothionein fraction is substituted by Cd.

The mutagenicity of organotin compounds as environmental pollutants

The mutagenicity of 14 organotin compounds which have been reported to be environmental pollutants, their environmental metabolites and inorganic tin (SnCl4) was studied. The experiments were carried out by a modification of the conventional Salmonella assay. Each tested chemical was removed by washing the tested strain with phosphate buffer, before the strain with top agar was poured onto minimal glucose agar. By this method, we were able to estimate the mutagenicity of organotin compounds which had antibacterial activity. It was apparent that mono-n-butyltin oxide, n-butyltin trichloride, di-n-butyltin dichloride, tri-n-butyltin chloride, bis-(tri-n-butyltin)-oxide and dimethyltin dichloride were mutagens on Salmonella typhimurium TA100 and bis-(tri-n-butyltin)-oxide showed the highest mutagenicity. With S. typhimurium TA98, di-n-butyltin dichloride was found to be a mutagen.

Isolation of substances from glossy privet (Ligustrum lucidum Ait.) inhibiting the mutagenicity of benzo[a]pyrene in bacteria

Methanol and hot-water extracts of glossy privet (Ligustrum lucidum Ait.) inhibited the mutagenic activity of benzo[a]pyrene in Salmonella typhimurium TA98 with S9 mix. The methanol extract was fractionated with ether and n-hexane. As the active components, oleanolic and ursolic acids were isolated, which were soluble in ether and insoluble in n-hexane. The hot-water extract was fractionated to water, 60% and 100% methanol fractions. Nuezhenide was isolated from the 60% methanol fraction as the active component.

The genotoxicity of organotin compounds in SOS chromotest and rec-assay.

In these days pollution by organotin compounds in the environment extends widely and effects on human health are feared. We studied the genotoxicity of various organotin compounds (butyltins, phenyltins, methyltins) and inorganic tin (SnCl4), which are present in the environment, with the SOS chromotest and the rec-assay. Mono-n-butyltin oxide, n-butyltin trichloride and di-n-butyltin dichloride showed high SOS-inducing potency in the SOS chromotest with Escherichia coli PQ37. Di-n-butyltin dichloride, tri-n-butyltin chloride, bis(tri-n-butyltin)oxide, dimethyltin dichloride and trimethyltin chloride were recognized as genotoxic chemicals by the rec-assay.

Cadmium-binding protein (metallothionein) in carp.

When carp (Cyprinus carpio) were exposed to 5 and 30 ppm Cd in the water, the contents of Cd-binding protein, which has low molecular weight, increased in the hepatopancreas, kidney, gills and gastrointestinal tract with the duration of exposure. This Cd-binding protein was purified from hepatopancreas, kidney, gills, and spleen of carp administered 2 mg/kg Cd (as CdCl2), intraperitoneally for 6 days. Two Cd-binding proteins were separated by DEAE-Sephadex A-25 column chromatography. These proteins had Cd-mercaptide bond, high cysteine contents (ca. 29-34%), but no aromatic amino acids or histidine. From these characteristics the Cd-binding proteins were identified as metallothionein. By using antiserum obtained from a rabbit to which carp hepatopancreas MT-II had been administered, immunological characteristics between hepatopancreas MT-I, II and kidney MT-II were studied, and a slight difference in antigenic determinant was observed among them. By immunological staining techniques with horseradish peroxidase, the localization of metallothionein was investigated. In the nontreated group, metallothionein was present in the acinar cells of hepatopancreas and renal convoluted tubules. In the Cd-treated group (2 mg/kg IP daily for 3 days), metallothionein was present in the nuclei, sinusoids, and extracellular space of hepatopancreas, in addition to the acinar cells. Carp were bred in 1 ppm Cd, 5 ppm Zn solution, and tap water for 14 days, following transfer to 15 ppm Cd solution, respectively. The survival ratio was the highest in the Zn group followed by Cd-treated and control groups. The metallothionein contents increased in hepatopancreas and kidney in the order: Zn greater than Cd greater than control group. ImagesFIGURE 5.FIGURE 6.

Formation of metallothionein in fish

1. Carp were bred in Cd solution, and each organ was homogenized and centrifuged at 105,000 g. The Cd content of the supernatant was much more than that of the precipitate. 2. In examining the metal form in the supernatant, the induction of metallothionein (MT) was found in carp exposed to Cd and Zn solutions and the presence of metallothionein was found in fish captured in Nagara river and breeding ponds. 3. Cd content in the metallothionein fraction of hepato-pancreas increased at the early stages of exposure, but Cd in the metallothionein fraction of the other organs increased as time proceeded. 4. In the tap water group, metallothionein in hepato-pancreas and kidney contained Cu, and increased with the induction of metallothionein in hepato-pancreas due to Zn-exposure.

Investigation of the hemolytic effects of various organophosphoric acid triesters (OPEs) and their structure‐activity relationship

The hemolytic effects of various organophosphonc acid triesters (OPEs) were investigated and they showed strong hemolyic toxicity except triethyl phosphate and tris(chloroethyl)phosphate. 2‐Ethylhexyl diphenyl phosphate (EHDP) showed the strongest toxicity. By quantitative structure‐activity relationship (QSAR) study, one‐parameter regression equation to estimate hemolysis was not obtained. But, two‐parameter regression equations were obtained which were enought to estimate EC50 and EC20. The correlation coefficients with the two‐parameter regression equations were 0.939 for log(l/EC50) and 0.946 for log(l/EC20).

Inhibitory action of peony root extract on the mutagenicity of benzo[a]pyrene

The inhibitory effects of peony root extract on the mutagenicity of benzo[a]pyrene (B [a]p) have been investigated in the Salmonella typhimurium reversion test. Four kinds of experiments were performed: direct chemical reaction (1) between peony root extract and B [a]p, and (2) between peony root extract and active metabolite(s) of B [a]p, (3) inhibition of metabolic processes of B[a]p with S9 mix, and (4) inhibition of activation on mutagenicity. Peony root extract interfered with the action of enzymes in the S9 mix, and inactivated the activity of B[a]p metabolites. The bio-antimutagenic effect was assayed by reversion in Salmonella typhimurium TA98 and TA100.

Mechanism of antimutagenicity of aquatic plant extracts against benzo[a]pyrene in the Salmonella assay

The mechanism of antimutagenicity of water extracts of grass-wrack pondweed (Potamogeton oxyphylus Miquel), curled pondweed (Potamogeton crispus L.) and smartweed (Polygonum hydropiper L.) towards benzo[a]pyrene mutagenicity in Salmonella typhimurium was investigated. The antimutagenic components in the aquatic plants were water-soluble, heat-resistant and had a high molecular weight; chlorophyll did not play an important role.