Isolation and characterization of senile amyloid--related antigenic substance (SASSAM) from mouse serum. Apo SASSAM is a low molecular weight apoprotein of high density lipoprotein.

Sera obtained from senescence-accelerated mouse (SAM) and normal mice contained a substance that reacted with antiserum raised against ASSAM, a novel senile amyloid fibril protein isolated from the liver of SAM. This physiological substance, termed "SASSAM" (serum ASSAM-related antigenic substance), migrated to the albumin/prealbumin region in immunoelectrophoresis and the precipitation line formed with anti-ASSAM antiserum was stained positively with both Amide Black 10 B and Oil Red O/Fat Red 7B solutions, thereby suggesting that SASSAM is an alpha lipoprotein. Using Sephadex G-200 gel chromatography, SASSAM was eluted as a high mol wt form of approximately 200,000 daltons. Fractionation of lipoprotein from normal mouse serum by preparative ultra-centrifugation disclosed that SASSAM was found mainly in high density lipoprotein, HDL (the density is between 1.063 and 1.21 g/cm3). The largest amount of SASSAM was found in the HDL2 fraction (the density is between 1.063 and 1.125) and in this fraction SAA was not detected. Furthermore, ASSAM immunoreactivity appeared in the low mol wt proteins (below 10,000 daltons) of apo HDL separated in the buffer containing 8 M urea through Sephadex G-200. In 8 M urea sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), the major components of apolipoproteins in this position, possibly corresponding to apo C proteins, have the same molecular weight, 5,200 daltons, as ASSAM and this component was labeled by anti-ASSAM antiserum after transfer to nitrocellulose paper.

Chromatographic analyses of 3-methylcholanthrene metabolism in adult and fetal mice and the occurrence of conjugating enzymes in the fetus.

Adult pregnant mice were given i.v. injections of (3H)3-methylcholanthrene (20 muCi in 1.1 mug/mouse) or (14C)3-methylcholanthrene (1.0 muCi in 48 mug/mouse). Ethanol extracts of their tissues were chromatographed on Sephadex LH-20. Three groups of 3-methylcholanthrene metabolites were obtained: one group as yet unidentified, one containing the hydrocarbon and hydroxylated derivatives, and a third consisting of conjugated metabolites from the treated adult mice and their fetuses. The conjugated metabolites in tissue and in bile were separated into two fractions; one was acted on by beta-glucuronidase and to a lesser extent by arylsulfatase, and the other was resistant to these enzymes but completely susceptible to acid hydrolysis. The hydrolysis resulted in altered chromatographic behavior characteristic of the hydroxy compounds, which also appear in tissue. The enzyme-resistant conjugates were predominant in brain, muscle, and lung, and the enzyme-labile conjugates were predominant in the kidney, liver, and bile of adult mice. These conjugated metabolites were also demonstrated in fetal mice; some appeared in the fetus as early as the thirteenth day of gestation, the most immature fetus so far examined. The resistant group was predominant in the early developmental stages of the fetus and the susceptible group was increased in the excretory organs such as the kidney, liver, and contents of the intestinal tract as the fetuses approached term. transplacental transfer of conjugated metabolites from the mother to the fetus did not take place, although the parent 3-methylcholanthrene and its nonconjugated metabolites were transferred. We therefore assume that drug-metabolizing enzymes, including hydroxylases and conjugases, are active in the fetal mouse tissues as well as in the adult.

Nacroautoradiographic assays in pregnant mice and their fetuses given N-acetyl-9-[14C]-2-aminofluorene and p-[14C]-dimethylaminoazobenzene.

The distribution of radioactivity from N-acetyl-9-[114C]-2-aminofluorene and p-[14C]-dimethylaminoazobenzene administered i.v. or p.o. to 18-day-pregnant mice was observed by means of autoradiography of longitudinal section of the whole mouse. Radioactive substances derived from these carcinogens passed through the placenta and were distributed in the fetal organs including the kidney and intestine. Column, thin-layer, and paper chromatography revealed the presence, in while fetuses as well as in maternal livers, of N-acety-9-[14C]-2-aminofluorene. N-hydroxy-N-acetyl-9-[14C]-2-aminofluorene, ring-hydroxy-N-acetyl-9-[14C]-2-aminofluorene, and p-[14C]-dimethylaminoazobenzene. These results establish that N-acetyl-9-[14C]-2-aminofluorene and [14C]-p-dimethylaminoazobenzene are transported, metabolized, and excreted in the mouse fetus.

Identification of benzo(a)pyrene metabolites by gas chromatograph-mass spectrometer.

A gas chromatograph-mass spectrometer was used in an attempt to achieve rapid separation and accurate identification of benzo(a)pyrene and its synthesized derivatives. All derivatives, after being trimethylsilylated, were developed on Dexsil-300 and OV-1 columns. The seven diols and four stereoisomeric 7,8,9,10-tetraols studied were separated successfully. The separation of the 12 phenols was unsatisfactory; 8- and 11-isomers appeared separately but the other 10 isomers made 3 peaks on the OV-1 column. Among the five derivatives reported to be present in animal tissues, 6-, 7-, and 9-phenols were separated, but 1- and 3-phenols were fused on the Dexsil-300 column. Quinones were converted to related dihydroxyl derivatives under silylation. The chromatographic separation of four of the six dihydroxyl derivatives was successful on the OV-1 column, but the 6,12- and 7,10-isomers remained in a single peak. The two diol-epoxides were unstable under silylation and therefore were detected by their breakdown products, 7,8,9-trihydroxy-7,8-dihydrobenzo(a)pyrene and tetraols. Data are listed on the mass spectra and retention times of all benzo(a)pyrene derivatives studied.

Spontaneous age-associated amyloidosis in senescence-accelerated mouse (SAM).

Morphological studies on spontaneous systemic amyloidosis were conducted on 222 senescence-accelerated mice (SAM) (P) and on 150 mice in the senescence-resistant series (R). Among the pathologic findings, amyloidosis showed the highest incidence in both SAM (79.7%) and R (32.7%). Although an extensive deposition of amyloid was evident in some aged mice in the R series, a more severe amyloidosis occurred with a higher incidence in the P series. There was a statistical significance between the incidence of amyloidosis and age, in both the P and R series. There were no differences in organ distribution and mode of amyloid deposition between the P and R series or between the sexes. In about 60% of the amyloid-positive cases in the 28 killed SAM and 7 mice in the R series, there were no signs of inflammation or neoplasm. The morphological features in SAM more closely resembled those seen in cases of murine spontaneous senile amyloidosis than the features seen in cases of experimentally induced amyloidosis. This model is expected to be a valuable tool with which to assess the relationship between amyloid deposition and the aging process or senescence, perhaps even cases of human senile amyloidosis.

A new murine model of accelerated senescence.

Five senescence-prone series of mice (P-1, P-2, P-3, P-4 and P-5) and three senescence-resistant series (R-1, R-2 and R-3) were obtained by continuous sister-brother breeding from five original litters of AKR mice with severe deterioration, and the three original litters of AKR mice with normal aging, respectively. A grading score system was adopted to evaluate the degree of senescence of these mice and a steady and irreversible increase in this grading score was seen with advancing age in both the R and P series. The high grading score in the P series was due to an earlier onset of loss of passivity and reactivity, loss of skin glossiness and increased coarseness, hair loss, periophthalmic lesions, increased lordokyphosis of the spine and a more marked increase in their severity with advancing age as compared to the R series. Among the P series, P-2 showed a 100% incidence of systemic amyloidosis after 6 months of age and P-3 a 70% incidence of cataract over 16 months of age. The life span in the P series was shortened by about 26% of that of the R series. In view of the evidence obtained from the survivors, the growth rate and Gompertz function, the aging pattern in the P series was considered to be an acceleration of senescence. The P series has been named "SAM" ("Senescence Accelerated Mouse").

[Five-week intravenous toxicity study of montirelin hydrate (NS-3) in rats followed by 4-week recovery test].

A repeated dose toxicity study of montirelin hydrate (NS-3), a new drug for the treatment of disturbance of consciousness, was conducted in Sprague-Dawley rats. Male and female rats were given the drug intravenously for 5 weeks at doses of 0 (control), 0.05, 0.5, 5 and 50 mg/kg. After discontinuation of the treatment, a 4-week recovery test was also conducted in the 0, 0.5 and 50 mg/kg groups. No deaths related to the treatment were observed. Tremor was seen in the 50 mg/kg group. Polyuria was observed in the 5 and 50 mg/kg groups. There were decreases in body weight gain in the 0.5 mg/kg group and over of males, and in food consumption in all male dose groups and increase in water consumption in the 5 and 50 mg/kg groups. In blood chemical examination, decrease in triglyceride was observed in the 5 and 50 mg/kg groups of males. Urinalysis showed increase in urine volume in the 0.5 mg/kg group and over. Ophthalmoscopic and hematologic examinations failed to show any abnormalities attributable to the treatment. Pathological examination disclosed serous cell hypertrophy of the submandibular gland in all dose groups and increase in its organ weight in the 0.5 mg/kg group and over. The changes mentioned above were satisfactorily reversible except for the serous cell hypertrophy of the submandibular gland in the 50 mg/kg group. The decrease in food consumption and serous cell hypertrophy of the submandibular gland in the 0.05 mg/kg group were considered to be of no toxicological significance. These results show that the NOAEL of montirelin hydrate is 0.05 mg/kg for 5-week repeated dose toxicity in rats.

[Reproductive and developmental toxicity studies of montirelin hydrate (1)--Fertility study in rats by intravenous administration].

A study of fertility and early embryonic development to implantation of montirelin hydrate (NS-3), a new drug for the treatment of disturbance of consciousness, was conducted in Sprague-Dawley rats. Male rats were given the drug intravenously from 63 days before mating to the end of mating period and female rats from 14 days before mating to day 7 of pregnancy; the dose levels for both males and females were 0 (control), 0.02, 1 and 50 mg/kg. The females were sacrificed on day 20 of pregnancy for examination of their fetuses. In the 50 mg/kg group, tremor, disappeared within some minutes, was observed during administration period in all animals. Food and water consumptions increased in the females and body weight gain was suppressed in the males. Moreover prolonged estrus cycle was observed at early period of the administration in the females, but it recovered at late period of the administration. However, there were no toxicities in the males and females in the 1 mg/kg or less groups. The drug had no adverse effects on reproductive function of the parent animals, or on development of fetuses. These results show that the NOAEL of montirelin hydrate are 1 mg/kg for general toxicity in parent animals, and 50 mg/kg for reproductive function of the parent animals and for development of fetuses.

[Reproductive and developmental toxicity studies of montirelin hydrate (2)--Teratogenicity and postnatal study in rats by intravenous administration].

A study of the effect of montirelin hydrate (NS-3), a new drug for the treatment of disturbance of consciousness, during the period of organogenesis was conducted in Sprague-Dawley rats. Female rats were given the drug intravenously at dose levels of 0 (control), 0.02, 1 and 50 mg/kg from day 7 to day 17 of pregnancy. Twenty-three or twenty-five female rats per dose level were sacrificed on day 20 of pregnancy for examination of their fetuses, and the pregnant rats (13-15 per dose levels) were allowed to deliver naturally for postnatal examination of their offspring. In the 1 or 50 mg/kg group, water consumption and the weights of adrenals of the dams increased and the weights of the thymus of the dams decreased. In addition, tremor, disappeared within some minutes, was observed from day 7 to day 16 of pregnancy in all dams given 50 mg/kg. Moreover, food consumption increased and the weights of the submaxillary glands of the dams given 50 mg/kg increased. The drug had no effect on the number of corpora lutea and implantations, on fetal mortality, on fetal body weights, on sex ratio, or on external, visceral and skeletal development of the fetuses. The drug also did not affect delivery. The drug did not have any adverse effects on the newborn such as the number of live newborns, birth index and body weights of live newborn, or on the postnatal development of the first generation offspring (F1) such as differentiation, functional development, emotionality, motor ability, learning ability or reproductive performance. The drug also had no adverse effects of the second generation offspring (F2). These results show that the NOAEL of montirelin hydrate are 0.02 mg/kg for general toxicity in mother animals, 50 mg/kg for pregnancy and delivery of mother animals and 50 mg/kg for development of their offspring.

[Reproductive and developmental toxicity studies of montirelin hydrate (3)--Teratogenicity study in rabbits by intravenous administration].

A study of the effect of montirelin hydrate (NS-3), a new drug for the treatment of disturbance of consciousness, during the period of organogenesis was conducted in New Zealand white rabbits. Female rabbits were given the drug intravenously at dose levels of 0 (control), 0.01, 0.1 and 1 mg/kg from day 6 to day 18 of pregnancy. Female rabbits were sacrificed on day 29 of pregnancy for examination of their fetuses. In the 0.1 mg/kg group, food consumption decreased slightly. In the 1 mg/kg group, tachypnea, salivation and rhinorrhea were observed, and body weight and food consumption decreased and water consumption increased. The drug had no effect on the number of corpora lutea and implantations, or on fetal mortality, on fetal body weights, on placental weight, on sex ratio, or on external, visceral and skeletal development of the fetuses. These results show that the NOAEL of montirelin hydrate are 0.1 mg/kg for general toxicity in mother animals, and 1 mg/kg for pregnancy of mother animals and for development of fetuses.

[Reproductive and developmental toxicity studies of prulifloxacin (NM441)(1)--A fertility study in rats by oral administration].

A study of fertility and fetal development of prulifloxacin, a new antibacterial agent, was conducted in Sprague-Dawley rats. Male rats were given the drug orally from 63 days before mating to the end of the mating period. Female rats were given from 14 days before mating to day 7 of pregnancy. The dose levels for both males and females were 0 (control), 30, 170 and 1000 mg/kg. The females were sacrificed on day 20 of pregnancy for examination of their fetuses. In the 170 mg/kg group, water consumption increased in the female rats, and food consumption decreased and the cecum increased in weight and was enlarged in the males and the females. In the 1000 mg/kg group, body weight gain was suppressed in the males, and food consumption decreased, water consumption increased and cecum increased in weight and enlarged in the males and the females. Moreover, this dose caused white spots and rough surface of the kidney in the males. Prulifloxacin had no adverse effects on reproductive function of the parent animals, and also on development of the fetuses. These results show that the NOAEL of prulifloxacin are 30 mg/kg for general toxicity in parent animals, 1000 mg/kg for reproductive function of parent animals and for development of fetuses.

[Reproductive and developmental toxicity studies of prulifloxacin (NM441)(2)--A teratogenicity study in rats by oral administration].

A study of the effect of prulifloxacin, a new antibacterial agent, during the period of organogenesis was conducted in Sprague-Dawley rats. Female rats were given prulifloxacin orally at dose levels of 0 (control), 30, 300, and 3000 mg/kg from day 7 to day 17 of pregnancy. Twenty-four or twenty-five female rats per dose level were sacrificed on day 20 of pregnancy for examination of their fetuses, and the pregnant rats (12-15 per dose levels) were allowed to deliver naturally for postnatal examination of their offspring. In the 300 or 3000 mg/kg groups, the food consumption decreased and water consumption increased in the dams. In the 3000 mg/kg group, the body weight gain was suppressed in the dams. In the 300 and 3000 mg/kg groups, the weights of cecum increased and the enlargement of cecum was observed. In the 3000 mg/kg group, white spots and rough surface of the kidney and renal tubular nephrosis with crystalline substance were observed and the weight of the kidney increased in the dams, and the body weight decreased and retarded ossifications in the fetuses. Prulifloxacin had no effect on the number of corpora lutea and implantations, or on fetal mortality, sex ratio, or external and visceral development of the fetuses. Prulifloxacin also did not affect delivery and the number of live newborns and birth index, or have any adverse effects on the postnatal development of the first generation offspring (F1) such as differentiation, functional development, emotionality, motor ability, learning ability or reproductive performance. Prulifloxacin also had no adverse effects of the second generation offspring (F2). These results show that the NOAEL of prulifloxacin are 30 mg/kg for general toxicity in mother animals, 3000 mg/kg for pregnancy and delivery of mother animals and 300 mg/kg for development of their offspring.

[Reproductive and developmental toxicity studies of prulifloxacin (NM441)(3)--A teratogenicity study in rabbits by oral administration].

A study of the effect of prulifloxacin, a new antibacterial agent, during the period of organogenesis was conducted in New Zealand White rabbits. Female rabbits were given prulifloxacin orally at dose levels of 0 (control), 10, 30 and 100 mg/kg from day 6 to 18 of pregnancy. Female rabbits were sacrificed on day 29 of pregnancy for examination of their fetuses. In the 30 mg/kg group, food and water consumption decreased. In the 100 mg/kg group, soft stool was observed and body weight gain, food consumption and water consumption decreased. Premature delivery (2/16) occurred and enlargement of cecum and increased weight of cecum were observed. The number of fetal death increased in the 100 mg/kg group. However, prulifloxacin had no effects on the number of corpora lutea, implantations and live fetuses, and on body weight, placental weight, sex ratio, and external, visceral and skeletal development of live fetuses. These results show that the NOAEL of prulifloxacin are 10 mg/kg for general toxicity in mother animals, 30 mg/kg for pregnancy of mother animals and for development of fetuses.

[Single dose toxicity studies of montirelin hydrate(NS-3) in mice, rats and dogs].

The single dose toxicity studies of montirelin hydrate (NS-3), a new drug for the treatment of disturbance of consciousness, were conducted in Slc:ddY mice, Slc:SD rats, and beagle dogs of both sexes. The drug was administered intravenously (i.v.) to mice, rats and dogs, and intramuscularly (i.m.) to mice and rats. The animals were observed for 14 days after administration. LD50 values were more than 500 mg/kg and 200 mg/kg in mice and rats, respectively, by the i.v. route, and more than 20 mg/kg in both animal species by the i.m. route. In dogs, the minimum lethal dose was more than 200 mg/kg by the i.v. route. Mice that received more than 125 mg/kg by the i.v. route showed tremor and a decrease in locomotor activity during administration and for 30 min thereafter. Mice that received more than 5 mg/kg by the i.m. route showed tremor 5 min after administration and for 2 hr thereafter. Rats that received more than 50 mg/kg by the i.v. route showed tremor, and those that received 200 mg/kg by the same route showed a decrease in locomotor activity and ataxic gait, during and immediately after administration. Rats that received more than 5 mg/kg by the i.m. route showed tremor, and those that received 20 mg/kg by the same route showed salivation 5 min after administration and for 30 min thereafter. Dogs that received more than 12.5 mg/kg by the i.v. route revealed excitement, biting, vocalization, mydriasis, salivation, urination, defecation, licking chops, vomiting, increase in heart rate, panting, hyperthermia, tremor and conjunctival injection during administration and for 6 hr thereafter. The body weight, food consumption and water consumption, and pathological findings showed no changes attributable to the dosing of montirelin hydrate in any animal.