The major toxicological problem today is to establish a useful method to cope with a low level and prolonged pollution. The toxicological method under such circumstances should include ecotoxicology and biotoxicology as well as classical toxicology, and is named as eco-biotoxicology. This method includes not only investigations on kinetics and levels of chemicals in the environment, human reaction and adaptation to chemicals or biophysiological and pathophysiological clarification of the effects, but also the establishment of countermeasures to prevent health hazards from chemicals, including biological monitoring and development of exposure parameters. The followings were the results recently obtained in our laboratory in support of the object of eco-biotoxicology. 1. Two low-molecular-weight chromium binding substances were purified from the liver of dogs. Chromium bound to them showed a lower toxicity and higher rates of urinary excretion than inorganic chromium compounds. A low-molecular-weight nickel binding substance was also purified from the liver of rats. Nickel bound to it showed a lower cytotoxity, but a higher malignant transformation induction of cultured cells than inorganic nickel compounds. These results indicate that chemical form of metals and binding protein in the body are important factors in their toxicity. 2. Triphenyl and tributyltins showed to inhibit insulin release from pancreas, collagen-induced aggregation of platelets, O₂^-production and chemotaxis of leukocytes, histamine release from mast cells, etc. A common mechanism seemed to be an inhibition of the stimulus-response coupling in the cell membrane. 3. Carcinogenic heterocyclic amines were measured in food and biological materials by a newly developed method. Their levels in plasma of uremic patients were higher than normal subjects and decreased by the treatment with hemodialysis. 4. Safety evaluation of bis (tri-n-butyltin) oxide (TBTO) in fishes was carried out according to a toxicological approach. Acceptable daily intake of TBTO in food was estimated to be 1.6 μg/kg/d.

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Recent progress in the metabolism of vitamin D and calcium is reviewed. Vitamin D is metabolized to 25-hydroxyvitamin D (25-OH-D) in the liver and subsequently to 1α, 25-dihydroxyvitamin D [1α, 25-(OH)₂D] or 24, 25-dihydroxyvitamin D [24, 25-(OH)₂D] in the kidney when plasma calcium concentrations are lower or higher than normal. 1α, 25-(OH)₂D, which is known as an active form of vitamin D, is transported to the target organs (intestine, bone and others) to show the physiological activity. The most important role of 1α, 25-(OH)₂D is to keep the calcium homeostasis by accerelating the intestinal calcium absorption and bone remodeling and the mechanism is discussed.

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In order to investigate the possible formation of chlorinated pyrene in the chlorination process for tap water, we carried out a model experiment on the chlorination reaction of pyrene (Py), and studied the chlorinated products and their stability by the light. The mutagenicities of the products were also investigated. The results obtained were as follows : 1) The main product was monochloropyrene (PyCl), when the chlorination reactions of Py were performed at pH 4.3, 20°C for 0.5-24 h in the dark with a hypochlorite solution containing 0.6 mg/l free chlorine (F-Cl₂). PyCl was hardly formed at pH 6.0 and above, and the formative amount of PyCl increased with the rise of reaction temperature. 2) Dichloropyrene (PyCl₂) was also formed, when the chlorination reactions of Py were performed at pH 3 with F-Cl₂ 0.6 mg/l and at pH 3-4.3 with F-Cl₂ 9 mg/l. 3) The concentrations of Py and PyCl decreased quickly in the presence of bromide ion under the acidic condition partly because monobromopyrene was produced. They decreased by the light irradiation. 4) PyCl and PyCl₂ were found to be mutagenic to both TA 98 and TA 100 strains in the presence of S-9 mix. From these results, and from pH value and residential time of treated water in a filter plant, the formation of the chlorinated products, such as PyCl and PyCl₂, seemed to be not likely to occur in the chlorination process for tap water.

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Identification of brand of hair care products (hair liquids, hair tonics, hair shampoos and hair rinses) by components remaining on human hair was investigated. They were identified by GC and GC-MS analyses after extraction with ether. Hair liquid of 57 brands produced by 17 companies were grouped into 21 classes on the basis of components remaining on human hair, and among them hair liquids of 11 brands were identified by peculiar components. Major components of hair liquids on human hair were able to be detected even 5 d after the treatment. However, remaining components of hair tonics were detected only 4 brands of 4 companies (in hair tonics of 51 brands of 14 companies). Three brands among them were identified even if used together with hair liquids because their remaining components did not overlap with those of hair liquids. Main components of those 4 brands hair tonics on human hair were able to be detected after 10 d. Remaining components were detected in only 1 brand of hair rinse among 20 brands of hair shampoo and hair rinse, and it was identified because the components differ from those of hair liquids and hair tonics. Therefore, the brands of hair tonics, hair rinse and some of hair liquids were identified if major components were detected from treated hairs.

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As a pretreatment in mutagenicity assays of non-volatile organic micropollutants in drinking water, the applicability of three methods of concentration (solvent extraction, XAD-2 column extraction and lyophilization) was investigated in conjunction with Ames Salmonella/microsome assays. Raw and tap water samples and ozonated and chlorinated humic samples used were assayed with strains TA 98, TA 100 and TA 104. When lyophilization was employed, the detection of the mutagenicity of organic micropollutants in tap water required at least 10000-fold concentration, and as a solvent of mutagenic materials in tap water, DMSO was more effective than distilled water. Of the three preconcentration methods, the acidic extracts in the ethyl ether extraction and the dichloromethane fractions in the XAD-2 column extraction were found to recover selectively mutagenic and non-volatile organic materials from all of raw water, tap water and chlorinated humic samples. In particular, the XAD-2-dichloromethane fraction was considered to recover quantitatively mutagenic products from chlorinated humic samples. These findings indicate that XAD-2 column extraction may be the most suitable preconcentration procedure for organic micropollutants in water.

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The present study was carried out to investigate the tissue distribution of toluene in rats with acute and subacute toluene intoxication. We used rats that had inhaled toluene vapor once for 30 min as a model of acute toluene intoxication and rats that had inhaled toluene vapor for 15 min per day for five days as a model of subacute toluene intoxication. Toluene levels in various tissues were measured. In the case of rats immediately after toluene inhalation, toluene levels in tissues were in the range of 71-587 ppm (μg/g tissue) for acute intoxication and in the range of 26-273 ppm for subacute intoxication. In particular, the liver and fatty tissue showed high toluene levels of over 450 ppm for acute intoxication and of over 200 ppm for subacute intoxication. When the rats were maintained for 17 h after exposure, fatty tissue and hair showed toluene levels similar to those immediately after inhalation. However, toluene in the liver decreased to about 1 ppm in the case of acute intoxication and to about 0.2 ppm in that of subacute intoxication. When rats with acute intoxication were killed immediately after toluene inhalation and kept for 17 h at room temperature, large amounts of toluene disappeared from the hair and spleen, but the other tissues showed levels similar to those immediately after inhalation. Repeated toluene inhalation did not lead to accumulation of toluene in any tissue.

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The simultaneous determination of 110 compounds including pesticides, hypnotics, local anesthetics, psychotropics, narcotics, etc., was examined by means of retention index on gas chromatography (GC). Temperature programmed GC using fused- silica capillary column CBP-1 (0.53 mm i. d.×12 m) was very effective in this study as compared with CBP-10 (0.33 mm i. d.×25 m) and some packed columns. All of the compounds except benzoximate, cloxazolam and oxazepam could be detected as good symmetrical peaks less than 100 minutes under the conditions of 120-250°C/(6°C/min) as oven temperature, 270°C as injection temperature, 0.2 kg/cm² as carrier gas (N₂) pressure (velocity, 90.9 cm/s) on CBP-1. A search window of these retention indices could be estimated about 20, then the discriminating powers of a combination of this data and TLC screening system mentioned earlier (Ref. 1), were 0.995, 1.00, 1.00, 0.996 and 1.00 in I, II, II', III and IV division, respectively.

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Thiabendazole (TBZ) suspended in olive oil or 3% gum arabic was given orally to pregnant ICR mice on d 15 or during d 6-15 of gestation at a dose of 1300 mg/kg/d, and TBZ including its metabolites, 5-hydroxythiabendazoles (5-OH TBZ and its conjugates) and the activities of the liver microsomal drug metabolizing enzymes were analyzed. The concentration of 5-OH TBZ was higher than that of its conjugates (glucuronide and sulfate) in the liver and placenta, whereas the levels of the conjugates were greater than that of the free form in the kidney 1 or 25 h after a single or repetitive administration of TBZ. Little sulfate of 5-OH TBZ was detected in the fetus. Aniline hydroxylase activity increased significantly after repetitive and a single administration (25 h after) of TBZ. The amount of cytochrome P-450 also increased significantly after repetitive administration of TBZ. Two vehicles (olive oil and 3% gum arabic) showed no significant difference in the data obtained above.

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Uptake and absorption of cadmium (Cd) through the airway were examined by determining the dose-dependent accumulation in the lung and its transfer to other organs after a single intratracheal instillation of cadmium chloride into rats. Male rats of the Wistar strain (10 weeks old ; body weight, 270±10 g (mean±S.D.)) were instilled with Cd dissolved in 0.4 ml saline at eight doses (0.313-40 μg Cd/rat) and killed two days later. Concentrations (μg/g tissue) and contents (μg/tissue) of Cd in the lung, liver, kidney and spleen were determined by flameless Zeeman atomic absorption spectrophotometry and/or inductively coupled argon plasma-atomic emission spectrometry (ICP). More than 90% of Cd were accumulated in the lung when the dose was lower than 2.5 μg Cd/rat, while the percentage of Cd accumulated in the lung decreased steeply at higher doses than 5 μg Cd/rat and the metal was transferred predominantly to the liver.

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Cadmium chloride dissolved in physiological saline (0.4 ml) was instilled intratracheally into male Wistar rats (10 weeks old ; body weight, approximately 270 g) at eight doses (40-0.313 μg Cd/rat). Concentrations of elements (calcium, copper, iron, magnesium, phosphorus, sulfur and zinc) in the lung were determined along with cadmium by inductively coupled argon plasma-atomic emission spectrometry (ICP) and/or flameless Zeeman atomic absorption spectrophotometry. The body weight decreased significantly, while the lung weight increased significantly at doses higher than 5.0 μg Cd/rat. Changes in lung weight and element concentrations suggested that lung edema was induced at a dose of as low as 2.5 μg Cd/rat. Distributions of copper and zinc in the supernatants of the lungs were determined on a gel permeation column by high performance liquid chromatography-ICP, indicating that superoxide dismutase in the lung decreased with a cadmium instillation at doses higher than 2.5 μg Cd/rat.

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