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When male Sprague-Dawley rats were administered d-alpha-tocopherol and butylated hydroxytoluene in the diet or intraperitoneally for 7 days, prolongations of prothrombin time and partial thromboplastin time were observed in those given both chemicals by both routes in a dose-dependent manner. However, intraperitoneal d-alpha-tocopherol was less toxic and the prothrombin and partial thromboplastin time indices were approx. 70% and approx. 60% in rats given 2.91 mmol/kg body weight daily. Rats given d-alpha-tocopherol in the diet at a daily dose of 2.31 mmol/kg body weight were approx. 13% and approx. 16% of the control, and in those dietary groups severe hemorrhages in epididymis and other organs were also observed. Plasma concentrations of total tocopherols were more increased by the dietary than the intraperitoneal route. These results suggest that the great difference in the hemorrhagic effect of d-alpha-tocopherol between dietary and intraperitoneal route administration may largely be due to the differing absorption rates of the drug by these two means.

o-Phenylphenol (OPP) and thiabendazole (TBZ), which are fungicides, have been approved as food additives in Japan. OPP induces urinary bladder tumor and renal lesions in rats but not in mice. TBZ has an enhancing effect on urinary bladder carcinogenesis induced by OPP in rats. The present study was performed to examine the combined effects of OPP and TBZ on mice. Groups of 20 male Crj: B6C3F1, mice were fed the basal diet (control, group C), diet containing 0.2% TBZ (group T), 0.65% OPP (group O-1), 1.3% OPP (group O-2), 2.6% OPP (group O-3), 0.65% OPP plus 0.2% TBZ (group OT-1), 1.3% OPP plus 0.2% TBZ (group OT-2) or 2.6% OPP plus 0.2% TBZ (group OT-3) for 52 weeks. The survival rates were 100% (20/20), 90% (18/20), 100% (20/20), 95% (19/20), 100% (20/20), 90% (18/20), 75% (15/20) and 70% (14/20) in group C, T, O-1, O-2, O-3, OT-1, OT-2 and OT-3, respectively. The treated groups suffered growth retardation throughout the study. The kidney weights showed a dose-related decrease in the O and OT groups. No treatment-related increase in tumor formation of any organ or tissue was observed. Dose-related increases were observed in the incidences of degenerative and necrotic changes in tubular epithelium and transitional cells of the kidney, and of anisonucleosis and pigmentation in liver cells in the O and OT groups. Under the experimental conditions employed, OPP plus TBZ induced no urinary bladder tumor in mice.

Phenacetin (acetophenetidine) is a broadly used analgesic and antipyretic drug, so the mutagenicity, clastogenicity or carcinogenicity is an important problem, which in experimental animals or cultured mammalian cells has been reported elsewhere (Ishidate and Odashima, 1977; Nakanishi et al., 1978; Isaka et al., 1979; Matsuoka et al., 1979). We examined the mutagenicity and clastogenicity of acetophenetidine (APT) and its derivatives, acetaminophen (AAP) and acetanilide (AAL), and perceived that AAP has a somewhat clastogenic action in vivo and in vitro, but APT and AAL have no such effect (Yoshida et al., 1980). No mutagenicity of any of the three chemicals assayed in vitro with cultured Chinese hamster cells (CHO-Ki cells) was recorded in our experimental system, in which the frequency of ouabain-resistant ceils was measured for cells treated with these compounds. However, an additional effect of acetaminophen (AAP) on the mutagenic and clastogenic action of a potent mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), was indicated in experiments with CHO-KI ceils in vitro. AAP, when it was added to the cultured ceils together with MNNG, enhanced the action of MNNG, and the levels of enhancement were dependent on the additional dosages of AAP. Fig. 1 shows the mutagenicity and cytotoxicity of MNNG (A) and AAP (B) on CHO-KI cells. One million cells were seeded in culture bottles, containing 10 ml of Ham FI2 medium supplemented with 10°/0 bovine serum, and incubated overnight at 37°C in a 5°/o CO2 incubator. About 16-18 h after the seeding, the cells were treated with various doses of MNNG or AAP in 10 p.! DMSO for 2 h. The medium was removed, cells were washed twice with fresh medium, and 10 ml fresh culture medium was put into each bottle. The treated cells were cultivated for a further 24

Guinea pig epidermal DNAase I was purified from an epidermal extract by a procedure including DEAE-cellulose chromatography, Sephadex G-100 gel filtration and Con A-Sepharose affinity chromatography. The purified enzyme contained no detectable activities of acid DNAase, alkaline RNAase, phosphodiesterase or acid or alkaline phosphatase, but was contaminated with acid RNAase activity. The molecular weight of the enzyme was estimated to be 33 000 by sucrose density gradient centrifugation and Sephadex G-100 gel filtration. Its isoelectric point is 5.2 +/- 0.1. The enzyme requires divalent cations and exhibits two pH optima that are dependent on divalent cations: in the presence of Mn2+, the optimum pH is about 7.5 in 50 mM Tris-HCl buffer and in the presence of Mn2+, the pH is 6.4 in 50 mM cacodylate-HCl buffer. The enzyme hydrolyzes native DNA about 6-times faster than denatured DNA, producing 5'-phosphoryl and 3'-hydroxyl terminated oligonucleotides with an average chain length of about eight nucleotides, and converts double-stranded and circular DNA to relaxed and linear forms. The enzyme is inhibited by G-actin and antiserum against bovine pancreatic DNAase A. Thus this enzyme is classified as DNAase I.

Infusion of 50 mU/100 g body wt of calcitonin induced a rapid and marked increase of cyclic AMP in calvaria as well as in plasma of thyroparathyroidectomized rats. These changes preceded the decrease in plasma calcium concentration seen after hormone administration. The possible relationship of this stimulation of cyclic AMP accumulation to the hypocalcemic effect of calcitonin was examined under several experimental settings. It was found that a dose of calcitonin sufficient to cause a significant decrease of plasma calcium failed to produce any detectable accumulation of cyclic AMP in calvaria, but when higher doses of calcitonin were given, there was an apparent correlation between the integrated change of cyclic AMP metabolism and the duration and magnitude of hypocalcemia. Experiments employing theophylline and imidazole, which alter cyclic AMP metabolism, led to an apparent dissociation between the hypocalcemic effect of calcitonin and its stimulation of cyclic AMP accumulation in bone. (Endocrinolo...

Nineteen healthy humans (2 men and 17 women) served as experimental subjects in 4 experiments using diets having different levels of leucine and also a valine-deficient diet. The effect of an excess intake of leucine, with and without addition of vitamin B-6, and the effect of a deficiency of valine on urinary excretions of N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, nicotinic acid, quinolinic acid, and 5-hydroxyindole acetic acid, and on the level of plasma amino acids were investigated. There was no effect of leucine on the excretion of these metabolites, but a marked decrease in the plasma (or serum) valine level was observed. The same decrease was seen when a valine-deficient diet was fed.

Orthophenylphenol (OPP), a widely used fungicide, induced ouabain-resistant (OuaR) mutants in a ultraviolet (UV)-sensitive human RSa cell strain and the frequencies increased in a dose-related fashion. OPP was a more potent mutagen than UV at doses related to equal survival. These results suggest that OPP has a mutagenic activity and that further experiments on this chemical are warranted.

An acid deoxyribonuclease has been purified from rat small intestinal mucosa by a procedure including ammonium sulfate fractionation, chromatographies on DEAE-cellulose, CM-cellulose and SE-Sephadex and finally isoelectric focusing. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed one major and two minor bands, and the enzyme activity corresponded to one of the minor bands. The enzyme preparation was free of contaminating DNase I, DNase III, alkaline RNase, acid and alkaline phosphatases and nonspecific phosphodiesterase, but slight activities of DNase IV and acid RNase were detected. The enzyme did not require divalent cations for activity, had a pH optimum of 4.5 in 0.33 M sodium acetate buffer, and had an optimum temperature of 50 to 60 degrees C when assayed for 30 min. The rate of hydrolysis of native DNA was about 2.5-fold faster than that observed with denatured DNA. Its molecular weight was found to be 9.0 +/- 0.1. The enzyme catalyzes the endonucleolytic cleavage of native and denatured DNA, yielding oligonucleotides which have an average chain length of about 7, and which contain 3'-phosphoryl termini. The mode of action of the enzyme is double-strand scission.

The effects of parathyroid hormone (PTH) on bone mineral mobilization are thought to involve the second messenger cyclic AMP. Intravenous administration of 10 U PTH induced rapid and marked accumulation of cyclic AMP in calvaria and plasma of thyroparathyroidectomized rats. The changes in cyclic AMP concentrations were not sustained, but returned to near-baseline values before the rise in plasma calcium concentration was manifest. Changes in the plasma levels of calcium and cyclic AMP in response to PTH infusion were demonstrated in thyroparathyroidectomized rats who were nephrectomized as well. In a series of experiments to clarify the association of cyclic AMP accumulation and calcium mobilization, it was found that small doses of PTH induced significant elevation of plasma calcium without stimulating an accumulation of cyclic AMP in calvaria, or plasma. Furthermore, in intact rats plasma calcium levels were elevated by endogenous PTH, provoked by EGTA induced hypocalcemia, without any demonstrable change of cyclic AMP metabolism in the skeletal tissue. At a higher dose of PTH, the magnitude of the integrated change of cyclic AMP metabolism in the skeletal tissue was well correlated to the amount of calcium mobilized. These observations indicate a coupling between enhanced production of cyclic AMP and calcium mobilization in the action of PTH on bone. They also suggest that either a minute change in cyclic AMP accumulation, which can not be detected due to a high basal level of this nucleotide in the tissue, or a change in metabolism of cyclic AMP in some particular cell compartment is an early event in PTH-induced calcium mobilization.

Porcine calcitonin (CT) was administered intravenously to 4 patients whose kidney function and plasma Ca and P levels were normal. A significant increase in urinary P excretion with an increase in urinary cyclic AMP (cAMP) excretion was observed. Plasma P and cAMP levels which were determined simultaneously did not show any significant change. Furthermore, plasma PTH levels determined by the radioimmunoassay revealed that the increased PTH levels provoked by the CT infusion was not responsible for an increase in urinary P or cAMP excretion, since no increase in plasma PTH levels was found during the period when an increase in urinary P or cAMP excretion was observed. In addition, the CT infusion caused an increase in urinary excretion of Ca, Na and Mg as well as P.Based on these data, it can be concluded that CT administered intravenously acts directly on the kidney, resulting in an increase in urinary P excretion which could be mediated through cAMP in the kidney.

When added to Eagle's Minimum Essential Medium supplemented with 10% bovine serum (MEM-10BS), 1mM cysteine was highly toxic to cultured cells. This toxicity was eliminated by (a) preincubation of the medium at 37 degrees C for 24 hr before use, or (b) presence of 5mM pyruvate. Similar results were obtained with freshly prepared CMRL 1066 supplemented with 10% bovine serum (CMRL-10BS), which contains 1.5 mM cysteine as an original ingredient. Medium L 15 supplemented with 10% bovine serum (L-10BS), which contains both 1 mM cysteine and 5 mM pyruvate, supported cell growth. On incubation of MEM-10BS supplemented with 1 mM cysteine (MEM-10BS-1CySH) or CMRL-10BS without cells for one day, the cysteine concentrations decreased to about one-tenth or less of the original concentrations. The cysteine concentration in L-10BS did not decrease so much on similar incubation. Pyruvate reduced the rate of disappearance of the cysteine in MEM-10BS-1CySH or CMRL-10BS as assayed with p-chloromercuribenzoate, although less than that in L-10BS. This effect of pyruvate was concentration dependent. These paradoxical effects of pyruvate on cysteine, i.e. the reduction of its cytotoxicity and the stabilization as an SH compound, are probably due to the formation of a dissociable complex between these two compounds, which is not cytotoxic and resistant to oxidation.

The level of cyclic AMP in various fractions of rat skeletal tissue was measured after in vitro or in vivo administration of parathyroid hormone and calcitonin. Incubations of bone fractions prepared from young (5 weeks of age) thyroparathyroidectomized rats revealed that both parathyroid hormone and calcitonin increased the cyclic AMP level in fractions of epiphysis, metaphysis and marrow cells. Cyclic AMP accumlation in incubated periosteum and diaphysis were induced solely by parathyroid hormone. In in vivo experiments, the cyclic AMP level in the tibia of the thyroparathyroidectomized rat was increased by infusion of either parathyroid hormone or calcitonin, and the simultaneous administration of each maximally effective dose of the two hormones exhibited an additive effect. Within 2 min, parathyroid hormone infusion caused an elevation of cyclic AMP content in periosteum and metaphysis. Rapid increase of cyclic AMP in the metaphysis was also induced by calcitonin, and the effect of the two hormones on cyclic AMP accumulation in this fraction was additive. Small but significant increase of cyclic AMP in the diaphysis was detected at 5 min after the administration of parathyroid hormone. Calcitonin infusion did not show any consistent effects on periosteum and diaphysis.

Both porcine calcitonin (CT) and bovine parathyroid hormone (PTH) increased the levels of cyclic adenosine 3',5'-monophosphate (cyclic AMP) in the whole kidney of rats in vivo andin the isolated renal cortical tubules of rats in vitro. Both hormones were without effect on cyclic AMP levels in the renal medullary tubules in vitro. Using doses that produce the maximal responses both in vivo and in vitro, the increments in cyclic AMP were greater with PTH than with CT, and additive effects were absent. Calcitonin and PTH producedan earlier peak in urinary excretion of cyclic AMP than in that of phosphate. These findings provide evidence that CT stimulates the production of cyclic AMP in the renal cortical tubules in vivo and in vitro, and suggest that there may exist a renal tubular cell population containing receptors for both hormones. The results are consistent with the contention that the phosphaturic effect of CT is mediated through the stimulation of adenyl cyclase-cyclic AMP system. (Endocrinology94:1...