Nobuhiro Konno

Long-term cadmium exposure induces anemia in rats through hypoinduction of erythropoietin in the kidneys

Abstract Cadmium (Cd), a highly toxic heavy metal, is distributed widely in the general environment of today. The characteristic clinical manifestations of chronic Cd intoxication include renal proximal tubular dysfunction, general osteomalacia with severe pains, and anemia. We have recently reported that the serum level of erythropoietin (EPO) remained low despite the severe anemia in patients with Itai-itai disease, the most severe form of chronic Cd intoxication. In order to prove that the anemia observed in chronic Cd intoxication arises from low production of EPO in the kidneys following the renal injury, we administered Cd to rats for a long period and performed the analysis of EPO mRNA inducibility in the kidneys. The rats administered Cd for 6 and 9 months showed anemia with low levels of plasma EPO as well as biochemical and histological renal tubular damage, and also hypoinduction of EPO mRNA in the kidneys. The results indicate that chronic Cd intoxication causes anemia by disturbing the EPO-production capacity of renal cells.

Subacute administration of tributyltin chloride modulates neurotransmitters and their metabolites in discrete brain regions of maternal mice and their F1 offspring

Tributyltin (TBT) compounds have been used as anti-fouling agents and the central nervous system is one of its target organs. TBT-induced modulations of neurotransmitters in the brains of adult mice have been reported. However, little is known about the developmental neurotoxicity of TBT. In this study, we evaluated the effects of TBT on neurotransmitters and their metabolites in discrete brain regions of female ICR mice and their offspring. Pregnant ICR mice were exposed to TBT chloride at concentrations of 0, 15 or 50 ppm in water or 125 ppm in food. Male offspring were sacrificed at one, two and three weeks after birth. The concentrations of norepinephrine, dopamine (DA), dihydoxyphenylacetic acid, homovanillic acid (HVA), serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) were determined in different brain regions by HPLC. All offspring from the 125 ppm group died immediately after birth. A significant decrease in the body weight of the TBT-treated F1 groups compared to the control group was observed in the first week. Significant increases compared to the controls were observed for the DA concentration in the striatum of the 50 ppm F1 group, and for the HVA concentration in the cerebrum and the 5-HT concentration in the medulla oblongata of the 15 and 50 ppm F1 groups in the third week. At three weeks of age, the neurotransmitters and their metabolites may be useful indexes for developmental neurotoxicity. For the dams, a significant decrease in the 5-HT concentration was observed in the cerebellum, medulla, midbrain and striatum of the 125 ppm group compared to the control group. A significant decrease in the 5-HIAA concentration was also observed in the cerebellum, midbrain and striatum of the dams in the 125 ppm group compared to the control. TBT may induce a decrease in the synthesis of 5-HT in the dams. The discrepancy between dams and offspring may be due to several factors such as age, dose, route, sex and pregnancy.

Delayed neurotoxicity of triphenyl phosphite in hens: Pharmacokinetic and biochemical studies

The organophosphorus compound, triphenyl phosphite (TPP), caused ataxia in chickens 8-14 days after single po or iv administration. The po and iv ED50 values were 1414 and 35.4 mg/kg, respectively. Chickens which developed ataxia lost 14.4 +/- 2.5% (mean +/- SEM, n = 14) of their initial weight at 28 days and the paralyzed birds showed a severe reduction of 29.3 +/- 2.9% (n = 13) of their initial weight at death or at 28 days after dosing. For the first 4-hr interval after iv injection of 50 mg/kg, the elimination of TPP from plasma consisted of at least two exponential phases; the half-lives of the first and second phases were approximately 30 and 60 min, respectively. When the birds received 100 mg/kg (iv) fatty tissue showed the highest TPP concentration, e.g., 215 micrograms/g fresh wt at 6 hr postdosing. The half-life was approximately 24 hr. Among neural tissues, the sciatic nerve had the highest concentration, followed by the spinal cord, the cerebellum, and the cerebrum. The red muscles, such as adductor magnus, contained about 4-30 times as much TPP as did the white muscles, such as biceps brachii, 6 hr after treatment. Time course effects of TPP treatment on mitochondrial enzymes in leg skeletal muscles were examined by treating hens with 50 mg/kg (iv) and euthanizing the birds at 6 hr to 8 days postdosing. The creatine kinase (CK) activities of the adductor and the soleus were significantly decreased at 2 (48 hr), 4, and 8 days, and at 4 and 8 days postdosing, respectively. Adductor magnus and soleus succinate dehydrogenase (SDH) activities were decreased markedly at 24 and 48 hr, and at 2 (48 hr), 4, and 8 days, respectively. Cytochrome oxidase (COD) activity in adductor magnus and soleus did not decrease during the time course. Biceps femoris CK, SDH, and COD activities were not affected by TPP treatment at this dosage. These results suggest that TPP administration affects the mitochondrial metabolism in skeletal muscle, especially red muscle of chickens.

Characterization of high-affinity binding sites for diisopropylfluorophosphate (DFP) from chicken spinal cord membranes☆

The delayed neurotoxic organophosphate [3H]diisopropylfluorophosphate ([3H]DFP) binds with high affinity to membrane-bound proteins from the chicken spinal cord. The DFP binding proteins were solubilized from membrane preparations, using a detergent (CHAPS). The protein(s) sites that labeled with a low concentration of [3H]DFP, e.g. 10(-10)-10(-9) M, were defined as the high-affinity binding sites. The density (or concentration) of the high-affinity binding sites in protein(s) was determined by the difference between total and non-specific binding. The high-affinity binding sites were saturable, and the maximal amount of binding sites was estimated at 400 fmol/mg protein. [3H]DFP binding to solubilized proteins was not completely reversible. Concentration-dependent curves suggested that the [3H]DFP binding sites differ from the active sites of acetylcholinesterase, butyrylcholinesterase, and neuropathy target esterase, as well as from muscarinic acetylcholine receptors. The amount of DFP binding sites after a neurotoxic dose of tri-o-cresyl phosphate (TOCP) decreased markedly in membrane preparations from the chicken spinal cord. These results indicate that a TOCP metabolite(s) interacts with the DFP binding sites in vivo. Gel filtration chromatography of the solubilized membranes indicated at least two major high-affinity DFP binding proteins with apparent molecular weights of 300 and 110 kDa. The DFP binding sites corresponding to the 110 kDa protein were insensitive to eserine, a potent anti-cholinesterase agent.

Effect of phenobarbital on the delayed neurotoxicity of leptophos in hens.

Intravenous administration of leptophos [O-(4-bromo-2,5-dichlorophenyl)O-methyl phenylphosphonothioate] caused a delayed neurotoxic effect in hens similar to that produced by oral and dermal administration. The intravenous (IV) ED50 for producing ataxia was estimated to be 21.6 mg/kg, less than one-tenth of the oral ED50 value. Pretreatment of the hens with the drug metabolizing enzyme inducer, phenobarbital (PB), caused a marked change in the biological half-life of leptophos in plasma. Plasma leptophos concentrations decreased in a first-order manner in control hens with a half-life of 36.0 min. In hens pretreated with PB, the elimination of leptophos from plasma consisted of at least two exponential phases, and the half-lives of the first and second phases were approximately 20 min and 140 min, respectively. When the birds received 40 mg/kg of leptophos IV, the incidence of ataxia was 8/18 (affected/tested) in the hens pretreated with PB and 19/19 in the hens without pretreatment. Phenobarbital significantly decreased the incidence of delayed neurotoxic signs (p < 0.01). The pretreatment of hens with PB resulted in a 59 to 69% decrease in the leptophos level in tissues including the brain and spinal cord after IV administration of 40 mg/kg. The results suggest that the protective effect of PB on delayed neurotoxicity is associated with a decreased concentration of leptophos in the target tissue(s).

The Effect of in Vitro and in Vivo Ethylenbis Dithiocarbamate Fungicides on NMDA Receptors in Rat Brain Membranes

To determine whether the ethlenbisdithiocarbamate fungicides, zineb, manzeb and maneb affect the N-methyl-D-aspartate (NMDA) receptor in rat brain membranes, we performed a binding assay using [3H]MK-801, a noncompetitive NMDA receptor antagonist. Displacement studies were conducted using well washed membranes to exclude the effect of endogenous acidic amino acids on the binding of [3H]MK-801. In both the presence or absence of added glutamate and glycine in the assay buffer, the dose-response curve indicated that zineb enhanced the binding in a concentration range of 100–500 μM. However, the displacement curves indicated that manzeb and maneb inhibited the binding in a concentration range of 10–500 μM. The addition of 50 μM glutamate and glycine to the assay medium increased binding by 5–20% above the control in a concentration range of 0.1–100 μM.No rats injected with zineb, manzeb, maneb (100 mg/kg, ip) showed any characteristic toxic signs or any significant weight changes within 24 hrs. Estimation of [3H]MK-801 binding to unwashed membranes from intoxicated rat brains revealed no marked change in Bmax or Kd values for 24 hrs following fungicide administration.