Toxicological studies using cultured mammalian cells are extremely useful and expected to be important alternatives to animal tests and experiments. There are various types of cultured cell experiments for various purposes, which need strict step-wise validation studies when used as the routine tests. The following test methods under validation studies are explained here ; direct cytotoxicity test, cytotoxicity test using cultured myotubes for intramuscular injection drugs, mouse lymphoma assay for gene mutation, in vitro cell transformation test for carcinogens and tumor promoters, and metabolic cooperation assay for tumor promoters. In order to achieve alternatives of animal experiments, researchers engaging in toxicological studies and recognizing their necessity are required to cooperate together for the further development of the experimental methods.

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The use of ion-selective electrodes for the determination of drug substances is reviewed. Although the primary emphasis is placed on drugs in the field of forensic chemistry, for example, paraquat, methamphetamine, amphetamine, and cocaine, other clinically important drugs such as procainamide, bretylium, disopyramide, and salicylate, as well as serotonin, a biogenic amine, are also discussed. Sensor membranes of these ion-selective electrodes can be prepared by incorporating a lipophilic ion-exchanger (or neutral carrier) and an appropriate membrane solvent in a poly (vinyl chloride) membrane matrix. In many cases, the combination of the ion-exchanger, sodium tetrakis [3, 5-bis (2-methoxyhexafluoro-2-propyl) phenyl] borate and the membrane solvent, 2-fluoro-2'-nitrodiphenyl ether afforded the most sensitive sensor membrane for drugs. In some cases, the choice of membrane solvent was important and tris (2-ethylhexyl) phosphate, tricresyl phosphate, and tetrakis (2-ethylhexyl) pyromellitate became excellent membrane solvents for making sensor membranes responding to serotonin, methamphetamine and cocaine, respectively. Neutral carriers may be useful to make highly selective electrodes to specific drugs, but have not been studied in detail. We discussed here amphetamine-selective and salicylate-selective electrodes using the neutral carriers, dicyclohexano-18-crown-6 and heptyl-4-trifluoroacetylbenzoate, respectively, as typical examples.

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The effects of variously subsituted pyridine and imidazole compounds and some of their chlorinated derivatives on the induction of hepatic microsomal heme oxygenase (HO) and cytochrome P450 (P450) were investigated in rats. 2-Benzylpyridine was able to induce HO to about 4.4-fold the control levels. 3-Benzylpyridine also increased HO to a lesser extent, but 4-benzylpyridine did not. Benzoylpyridines, phenylpyridines and diphenylmethylpyridines also produced a similar regio-differential induction of HO to benzylpyridines. Of the pyridine compounds examined, 2-phenylpyridine was the most potent inducer of HO. These substituted pyridines also increased the hepatic P450 content by inversely relating to the magnitudes of their abilities to induce HO. These results indicate that the substituted pyridines which have some lipophilic moieties, such as phenyl-, benzyl-, benzoyl- and diphenylmethyl-moieties, bound at the 2-position, but not at the 4-position of the pyridine ring, could induce HO. The substituted imidazoles generally produced a potent induction of P450, and some of them also exhibited HO induction similar to those of the corresponding pyridine compounds. Both 2-(4-chlorobenzyl)-pyridine and 2-(3, 4-dichlorobenzyl) imidazole were more potent inducers of HO than their dechlorinated parent compounds. All of these findings suggest that the substituted pyridine and imidazole compounds produce HO and P450 induction in a regio-related manner with lipophilic moieties, and that the addition of chlorine atom (s) may alter the magnitude of induction of HO in rat liver.

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Cytotoxic and lipid peroxidative effects of 4 major trihalomethanes (chloroform, CHCl₃ ; bromodichloromethane, BrCHCl₂ ; chlorodibromomethane, ClCHBr₂ ; bromoform, CHBr₃) were examined in isolated rat hepatocytes. The phospholipid hydroperoxides, phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH), were determined by the HPLC-chemiluminescence detection (CL-HPLC) method which is sensitive and specific for lipid hydroperoxide. Severe cytotoxicity was observed when the cells were cultured with 50 mM of CHCl₃, 20 mM of BrCHCl₂, 10 mM of ClCHBr₂ and 10 mM of CHBr₃, respectively. At these concentrations, cytotoxicity was observed after 10 min or more incubation with each 4 trihalomethane. The cellular lipid hydroperoxide was remarkably increased with the cytotoxicity by BrCHCl₂ and the other three trihalomethanes induced slight lipid peroxidation. The cytotoxicity was not effectively prevented by α-tocopherol, though the lipid peroxidation was appreciably prevented. These results suggested that trihalomethane induces lipid peroxidation in isolated rat hepatocytes, but that this may not be the main cause of the cytotoxicity.

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Subcutaneous injection (s.c.) of potassium cyanide (8 or 9 mg/kg) caused tonic and clonic seizures in 80 to 95% of the treated mice. However, the incidence of seizures was significantly reduced by intracerebroventricular preinjection of carbetapentane (50 nmol/brain), an inhibitor of glutamate release, or by subcutaneous preinjection of melatonin (20 mg/kg), a potent free radical scavenger. Potassium cyanide (8 mg/kg, s.c.) increased lipid peroxidation in the brain of mice 2.6-fold. The lipid peroxidation was completely abolished when tonic and clonic seizures were prevented by preadministration of melatonin (20 mg/kg). Furthermore, mortality elicited by potassium cyanide (8 or 9 mg/kg, s.c.) was also reduced by pretreatment with carbetapentane or melatonin. These results suggest that free radical formation and an increase in the release of glutamate may contribute in part to the development of neurotoxicity induced by potassium cyanide in mice.

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The early testicular damage after acute cadmium (Cd) exposure and the prevention of Cd-induced testicular toxicity by the oral administration of disulfiram (DSF) at various times after Cd exposure in rats were studied. Although moderate enlargement in interstitial tissues was observed at the first 3 h after Cd injection, no morphological changes in the inside of seminiferous tubules were observed. At 6 h after Cd exposure, damage of both interstitial tissues and seminiferous tubules was observed. Rats received an intraperitoneal injection of DSF (0.5 mmol/kg) or an oral administration of DSF (0.5, 1.0 or 2.0 mmol/kg) 0.5, 1, 3 or 6 h after the subcutaneous injection of CdCl₂ (26.7μmol (3 mg) Cd/kg). The oral treatment with DSF at dose levels of 0.5 mmol/kg (0.5 or 1 h), 1.0 mmol/kg (3 h), or 2.0 mmol/kg (3 h) prevented the increase in testicular lipid peroxidation and calcium and Cd concentrations and the decrease in testicular weight, which were observed at 7 d after Cd injection, as well as by the injection of DSF at 0.5 mmol/kg (0.5 h). The results at 59 d after Cd injection indicated that the testicular Cd concentrations and testicular weight after DSF treatment were changed in sterile rats, but not in fertile animals. These results indicate that Cd-induced testicular toxicity occurs 3-6 h after Cd injection, and that more effective chelation therapy with DSF for testicular toxicity by Cd is considered to arise from the oral administration of DSF at dose levels of more than 1.0 mmol/kg within 3 h after acute Cd exposure in rats.

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A gas chromatographic method using an electron capture detector is described for the determination of aldehydes in indoor air samples. It consists of the following three procedures ; (1) collection of aldehydes as O-(2, 3, 4, 5, 6-pentafluorobenzyl) hydroxylamine (PFBOA) derivatives on a 5% H₃PO₄-silica gel sorbent, (2) desorption of their aldehydes from the sorbent with a benzene solution, and (3) quantitative determination of the aldehyde derivatives by GC equipped with an electron capture detector. The recoveries of aldehydes from the air samples through the entire analytical procedures amounted to more than 90%. The calibration curves for fluoro derivatives of aldehydes were linear in the range from 0 to 200 pg per milliliter of benzene for each free aldehyde. The lower detection limits for formaldehyde and acetaldehyde were 2 and 3μg/m³, respectively. This method has been applied to aldehydes in indoor air samples. Formaldehyde and acetaldehyde were found in the air samples in the levels of 4.7-246 and 4.4-24.3μg/m³, respectively. The presence of vareraldehyde and capronaldehyde was also revealed in the indoor air samples.

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Keto-carboxylic acids will be important decomposition intermediates when ketones or fatty acids contained in water are treated with ozone. In this report 5 mM keto acids in the 0.05 M phosphate buffer (pH 7.0) were treated with UV irradiation alone, ozone alone or ozone + UV irradiation, and their decomposition products and the degradation pathway were investigated. The degradation pathways of keto-dicarboxylic acids were considered to produce dicarboxylic acid by oxidative decarboxylation at 1-position carbonyl group, α-keto-dicarboxylic acid having one carbon atom decreased by oxidative decarboxylation at the other terminal carbonyl group, and glyoxylic acid and dicarboxylic acid by releasing an α-keto-carboxylic acid group. And the main degradation pathway of keto-monocarboxylic acids containing a carbonyl group at α-position was considered to produce a monocarboxylic acid having one carbon atom decreased by oxidative decarboxylation. Other pathways were involved in the production of glyoxylic acid and monocarboxylic acid by releasing α-keto-carboxylic acid group, and keto-dicarboxylic acids by oxidation at a terminal methyl group. The degradation pathways of keto-monocarboxylic acids having a ketone group except α-position were considered to release a longer side-chain part prior to the other shorter chain. The released chains were considered to produce monocarboxylic acids or dicarboxylic acids. These intermediate products will be decomposed gradually to CO₂ and H₂O. TOC (total organic carbon) removal (%) was very high by ozone + UV irradiation. By the way, keto acids themselves by UV irradiation alone were decomposed some, because they has UV absorption spectrum about 254 nm which is the main irradiation wavelength of low presser mercury lump. But the TOC removal (%) by UV irradiation alone or O₃ alone was very low, because the decomposition products could not be decomposed by each treatment. These results will be utilized as important data when various ketones in the water are treated with O₃ and UV.

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An analytical procedure was developed for the determination of total bromide residues in crude drugs and their preparations, using modified X-ray fluorescence spectrometry. A 0.26 g of powdered sample mixed with 2.34 g of cellulose powder as vehicles was applied to a 40 mm i.d. aluminum ring, then pressed for one minute and set in spectrometer. The limit of the determination was 1μg/g (10μg/g as the original sample). Using the described method, cellulose powder as vehicles offers an extremely good property for mixing samples and less interference of sample matrix. We were able to eliminate the previous procedure, which is necessary to prepare standards for each part of sample due to being liable to variation. The modified X-ray method is advantageous for simple and high speed measurements and could be applied for various kinds of crude drugs and their preparations.

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A thiamin diphosphate (TDP) kinase (EC 2.7.4.15), which catalyzes the synthesis of thiamin triphosphate (TTP) from TDP, was found in erythrocytes after the freezing and thawing of human blood. The enzyme was biochemically distinct from the kinase previously reported. The enzyme activity was accelerated by Fe²⁺ and Fe³⁺ and inhibited by trichloroacetic acid, SH-blocking reagents and EDTA. It was suggested that the enzyme would have a sulfhydryl group at the active site, and requires certain cofactors for its activity.

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As a part of a series of studies to clarify the routes of human exposure and the environmental behaviour of termiticides, the analyses of 2, 3, 3, 3, 2', 3', 3', 3'-octachlorodipropylether (an organochlorine synergist, S-421) levels in surface water, sediments and rain were carried out. Surface water and sediments were collected from Osaka bay and three rivers near Osaka district. Rain was sampled at the roof of our institute in Osaka. S-421 was detected in all samples of the surface water and the sediments analyzed to the extent of 1.6 to 11.7 ng/l, and 1.5 to 3.2 ng/g, respectively. S-421 was detected in 9 of 13 samples of rain analyzed to the extent of 1.0 to 3.7 ng/l. Rain contained HCHs in the low ppt range.

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