The concentration of acetylcholine (ACh) in rat jejunum that had been homogenized with an ultra-high-speed homogenizer (Biotron<TM>) was significantly higher than that in jejunum homogenized with a glass homogenizer. Rats were injected once or repeatedly for 10 days with a muscarinic agonist, pilocarpine (1 mg/kg), or a muscarinic antagonist, scopolamine (5 mg/kg). Animals were killed 20 min or 24 hr after single or consecutive injections, respectively, for determinations of cholinergic activities in the jejunum. Single treatment: Pilocarpine did not cause significant changes in the level of ACh, the activity of acetylcholinesterase (AChE), the binding of [3H] quinuclidinyl benzilate (QNB) or the contractile responses to ACh. Scopolamine reduced the level of ACh and binding of [3H]QNB without inducing significant changes in the activity of AChE and the contractile response. Consecutive treatment: Pilocarpine reduced the binding of [3H]QNB by changing the value of Bmax and reduced the contractile response without affecting the level of ACh or the activity of AChE. Scopolamine increased the binding of [3H]QNB without any effects on the level of ACh, the activity of AChE or the contractile response. In summary, it is possible to determine the level of ACh in a tissue as hard as intestine by homogenization with a Biotron<TM> and to assess the cholinergic situation in the intestine of animals that have been poisoned with various agents by estimating cholinergic activities.
A confirmatory familiarization study of the Combined Repeat Dose and Reproductive/Developmental Toxicity Screening (ReproTox) test protocol proposed by the Organization for Economic Cooperation and Development (OECD) was performed using nitrobenzene, a testicular toxicant. The agent was given daily by gavage to groups of 10 male and 10 female Sprague-Dawley rats at doses of 100, 60, 20 and 0 mg/kg body weight. Some of the high dose animals exhibited neurological signs, and two males and 9 females died. Hemolytic anemia due to methemoglobin formation was evident in treated males. Histopathologically, treated males showed atrophy of seminiferous tubules of the testis, reactive changes secondary to hemolytic anemia in the hematopoietic organs, and hepatocellular swelling. Cerebral gliosis was observed in middle and high dose males. Male fertility was not affected. The body weights of pups from treated dams were lowered, and their postnatal loss was increased. Most of the known toxicological properties of this chemical was demonstrated in the present study, with the exception of reduced fertility. Therefore, the ReproTox protocol was concluded as being useful as a screening test of existing high production volume chemicals. It should be noted that while the reproductive toxicity test alone is insensitive for detection of male fertility disturbances associated with testicular toxicity, the latter easily be distinguished on morphological grounds.
Rats on day 21 of pregnancy received a subcutaneous injection of Paraquat (2, 7 or 25 mg/kg). The inner diameter of the ductus arteriosus (DA) of their fetuses was measured using the whole-body freezing method 1, 3, 6 and 24 hours after injection. The 7 mg/kg dose group showed a significant constriction of the DA 3 hours after paraquat treatment. The 25 mg/kg dose group showed a significant constriction 3 and 6 hours after treatment. The constrictive effect was dose-dependent, that is, the magnitude of constriction observed 3 hours after paraquat injection was greater in the 25mg/kg dose group than in the 7 mg/kg dose group. The 2 mg/kg dose group showed no significant change in the inner diameter of the DA at any point of measurement (1, 3, 6 and 24 hours after injection), compared to the saline treated controls. These results indicate that paraquat induces constriction of the fetal rat DA in a dose-dependent manner when administered in late stages of gestation, and that this effect of paraquat is not seen at a dose level of 2 mg/kg.
The occurrence of an autoantibody to protein disulfide isomerase (PDI) in rats after administration of various hepatotoxic drugs was investigated by immunoblotting and radioimmunoassay. An anti-PDI autoantibody was detected with high frequency in rats treated with D-galactosamine, acetaminophen with diethylmaleate, and carbon tetrachloride with diethylmaleate. The antibody-positive rate was relatively low in the groups of rats given carbon tetrachloride, acetaminophen or DL-ethionine alone. The anti-PDI antibody was not detected in rats treated with diethylmaleate alone. Although the mechanism of the production of the anti-PDI autoantibody is unclear, the occurrence of anti-PDI antibody correlated with high serum GPT activities. It is suggested that the autoantibody plays an important role in the development and persistence of drug-induced hepatitis.
The occurrence of autoantibody to protein disulfide isomerase (PDI) was examined in sera from patients with alcoholic liver disease, liver cirrhosis, systemic lupus erythmatosus (SLE) and cancer. Judging by results of Western blotting using a purified preparation of PDI, the anti-PDI antibody was hardly seen in most sera from healthy controls but was raised in patients with alcoholic liver disease, liver cirrhosis, SLE and liver cancer. When the antibody titer to PDI was measured by radioactivity in a radioimmunoassay, positive values (cut off value; mean+2SD=256 cpm) were seen in 46% (11/24) of the patients with moderate alcoholic liver disease (mean radioactivity, 339 cpm) and in 54% (7/13) of those with severe alcoholic liver disease (mean radioactivity, 664 cpm). Only 8% (4/48) of the healthy controls were positive (mean radioactivity, 50 cpm). Four of six patients with hepatoma were positive, while none of the seven patients with non-hepatic cancer were positive. The positive proportions in the patients with liver cirrhosis and SLE were 64% (9/14) and 40% (4/10), respectively. These results suggest that the occurrence of anti-PDI antibody would play an immunological role in the progression of hepatic disorders.
Newborn rats delivered by caesarean section were given subcutaneously verapamil hydrochloride (VER), a Ca-channel blocker, (1) immediately or (2) 180 min after delivery. The diameter of the ductus arteriosus (DA) of the newborn pups was calibrated at 30, 60 and 90 min after the VER-administration. (1) The DA calibers of the pups given 0.1, 1.0 mg/kg VER immediately after delivery remained significantly larger than those of control pups in a dose-dependent manner for 30 min after treatment. In the 1 mg/kg group, the enlargement of the DA was prolonged unti1 90 min after treatment. (2) In untreated pups, the DA completely closed by 180 min after caesarean delivery. The closed DA was not affected after 1 mg/kg VER was given at 3 hours after delivery. It was concluded that, VER inhibits the spontaneous constriction of the DA, suggesting that the increase of intracellular Ca<2+> concentration may play an important role on the spontaneous closure of the DA in newborn rats.