Interindividual variability in the expression of surfactant protein A and B in the human lung during development.

The surfactant complex, thanks to its multiple actions including decrease of surface- tension and antimicrobial activity, plays a fundamental role in newborn survival, lowering the risk of respiratory distress syndrome. The aim of this work was to determine if the synthesis of two surfactant proteins (SP), SPA and pro-SPB, shows some inter-individual variability during lung development in the intrauterine life. Immunoreactivity for SPA and pro-SPB was investigated in the lungs of  40 subjects, including 15 fetuses, ranging from 14 to 22 weeks of gestation, and 25 neonates, from 24 to 41 weeks. Lung samples were formalin fixed, paraffin-embedded and routinely processed. SPA and pro-SPB were detected utilizing commercial antibodies.  A semi-quantitative grading system (1 to 4) was applied, based on the number of reactive cells and the intensity of immunostaining. Surfactant protein immunostaining was found in  three compartments: bronchi, bronchioles and alveoli, starting from 14 weeks of gestation in the bronchial epithelium and from the 21st week in the alveolar spaces. Differences were found regarding SPA and pro-SPB expression in the vast majority of subjects: in some lungs, SPA was more expressed whereas in others pro-SPB showed an higher degree of immunoreactivity. The expression of both surfactant proteins was not strictly correlated with gestational age. Whereas the highest levels of reactivity were detected in at term neonates, on the other hand one case with grade 3 was detected at 22 weeks and one negative case for both proteins was observed at 31 weeks. Our data clearly show a marked inter-individual variability regarding the production of SPA and pro-SPB and suggest the existence of other epigenetic factors, acting during gestation, that might influence surfactant production and, consequently, the survival potential of  neonates at birth.

A novel pathway for formation of thiol metabolites and cysteine conjugates from cysteine conjugate sulphoxides.

p-Bromothiophenol and S-(p-bromophenyl)-L-cysteine were formed enzymatically from S-(p-bromophenyl)-L-cysteine sulphoxide in the in vitro systems with isolated rat hepatocytes or purified cysteine conjugate beta-lyases. Isotope dilution study with non-radiolabelled carrier of each product suggested the initial liberation of the thiol and subsequent formation of the cysteine conjugate. C-S bond cleavage pathway to liberate sulphenic acid and thiol are postulated to play an important role in in vivo generation of toxic intermediates and products from cysteine conjugates.

Purification and characterization of 3-mercaptolactic acid S-conjugate oxidases.

Two enzymes catalysing the oxidative formation of 3-mercaptopyruvic acid S-conjugates from L-3-mercaptolactic acid S-conjugates were purified to apparent homogeneity from rat liver cytosol. The two enzymes, tentatively designated MLO-I and MLO-II, showed a molecular mass of 160 and 250 kDa and were composed of four and six subunits of 41 and 39 kDa, respectively. Both enzymes possessed flavin mononucleotide as prosthetic group and oxidized several aromatic and aliphatic S-substituted L-3-mercaptolactic acids as well as alpha-hydroxy acids such as L-3-phenyllactic acid and L-2-hydroxyisocaproic acid. Glycolic acid and 3-(4-hydroxyphenyl)-lactic acid were the specific substrates for MLO-I and MLO-II, respectively. Neither of the enzymes oxidized beta- and gamma-hydroxy acids such as 3- and 4-hydroxybutyric acid. 2-Hydroxyisobutyric acid, ethyl-2-hydroxybutyrate, malic acid, 1-butanol, benzyl alcohol and L-leucine did not act as substrates for the enzymes. MLO-I and MLO-II exerted their maximum activities around pH 5.5 with Km of 0.5 and 0.25 mM and Vmax of 0.9 and 0.2 mumol/min/mg, respectively, when S-(4-bromophenyl)-3-thiolactic acid was used as substrate. MLO-I was inhibited by sulphydryl-modifying agents, while MLO-II was not. Both enzymes were strongly inhibited by divalent metal ions. These results indicate that MLO-I and MLO-II are different from L-amino acid oxidase (EC 1.4.3.2), malate oxidase (EC 1.1.3.3), L-alpha-hydroxy acid oxidase (EC 1.1.3.15) and glycolate oxidase (EC 1.1.3.1). The present enzymes are likely to be involved in the formation of cysteine conjugates from L-3-mercaptolactic acid S-conjugates in conjunction with cysteine conjugate aminotransferases.

Purification and characterization of 3-mercaptopyruvic acid S-conjugate reductases.

Three kinds of 3-mercaptopyruvic acid S-conjugate reductase (MPR-I, MPR-II and MPR-III) were purified from rat liver cytosol. These enzymes reduced 3-mercaptopyruvic acid S-conjugates derived from cysteine conjugates and some endogenous alpha-keto acids to the corresponding alpha-hydroxy acids in the presence of either NADH (for MPR-I and MPR-II) or NADPH (MPR-III), while simple aldehydes or ketones did not significantly induce substrate activity. The molecular weight of the present enzymes was about 80 kDa composed of two subunits of the same molecular weight. Km values of MPR-I, MPR-II and MPR-III were 0.38, 0.06 and 0.29 mM for S-(4-bromophenyl)-3-thiopyruvic acid, respectively, and 0.15 mM for NADH (MPR-I, MPR-II) and NADPH (MPR-III). Vmax values of MPR-I, MPR-II and MPR-III for this substrate were 5.3, 20 and 13 nmol/min/mg, respectively. The sulphydryl-modifying agents inhibited the enzyme activities of all the three reductases. Based on the properties including substrate selectivity for alpha-keto acids derived from aromatic amino acids, we assumed that MPR-II and aromatic alpha-keto acid reductase are the same enzyme, while enzymes similar to MPR-I and MPR-III have not been reported. From the viewpoints of metabolism of xenobiotics, these enzymes are likely to be important in biotransformation of cysteine conjugates to 3-mercaptolactic acid S-conjugates.

Carcinogenicity study of gamma-oryzanol in F344 rats.

The carcinogenic potential of gamma-oryzanol, a drug mainly used for the treatment of hyperlipidaemia, was studied in F344 rats. Groups of 50 males and 50 females were fed a diet containing 0 (control), 200, 600 or 2000 mg gamma-oryzanol/kg body weight/day for 2 yr. Although females in the highest dose group (2000 mg/kg body weight) showed a slight decrease in body weight at 104 wk, there were no treatment-related changes in general condition, food consumption, mortality, organ weight or haematology. Histopathological examination showed various tumours in all groups, including the control group. In the control and 2000-mg/kg groups, high tumour incidences were observed in the testes, pituitary and thyroid of males, and in the pituitary, uterus and mammary gland of females; however, there was no significant increase in the incidence of any tumours between the control and the 2000-mg/kg groups. The findings indicate that under the experimental conditions described gamma-oryzanol was not carcinogenic in F344 rats.

[Studies on antigenicity of difluprednate--antigenicity of difluprednate, phototoxicity and photocontact sensitivity of difluprednate ointment and cream].

The antigenicity of difluprednate, phototoxicity and photocontact sensitivity of difluprednate ointment and cream were studied in guinea pigs and mice. The results in this study were as follows. Guinea pigs immunized with difluprednate emulsified with Freund's complete adjuvant did not exhibit the systemic anaphylaxis and the delayed type hypersensitivity by the elicitation with either difluprednate alone or difluprednate-BSA conjugate. The antisera obtained from these guinea pigs did not show the positive response in the homologous 4-hour PCA reaction and the passive hemagglutination test against the challenge of the same antigens described above. In the maximization test, guinea pigs immunized with difluprednate did not show the contact sensitivity when elicited with difluprednate suspended in saline. Guinea pigs applied difluprednate ointment did not show positive contact sensitivity by the elicitation with difluprednate ointment and ointment base. The antisera obtained from mice immunized with difluprednate and difluprednate-OVA conjugate absorbed to Al (OH)3 gel did not give the positive response in the 72-hour PCA reaction against the challenge of either difluprednte alone or difluprednate-BSA conjugate. In the phototoxicity and photocontact sensitivity test, 0.05% difluprednate ointment and 0.05% difluprednate cream did not show positive reactions in guinea pigs, although very slight erythema was caused by the primary irritancy of cream base, with or without irradiation of ultraviolet ray (320-400 nm).

[Absorption, distribution, metabolism and excretion of netilmicin in rats (I). Metabolic fate of netilmicin after a single dose (author's transl)].

The absorption, distribution and excretion of netilmicin (NTL) in rats were examined by the microbiological assay method. Plasma level and urinary excretion of NTL in rats after intramuscular administration were compared with those of 5 other aminoglycoside antibiotics, gentamicin, sisomicin, dibekacin, amikacin and tobramycin. 1. After intravenous administration, plasma level of NTL declined rapidly with a half-life of about 18 minutes. In the cases of intramuscular, intraperitoneal and subcutaneous administrations, plasma levels reached maximum at 15 minutes and declined with similar half-life as intravenous administration. 2. When 20 mg/kg of NTL was administered intramuscularly, NTL was highly distributed into the kidney, while poorly into the brain. The disappearance rates of NTL from the tissues except the kidney were almost same as that from the plasma. Concentration of NTL in kidney was about 3 times higher than that in plasma at 15 minutes after administration and declined with a half-life of about 7 days. 3. NTL excreted in the bile within 24 hours was only 0.2% of the dose administered (20 mg/kg, i.m.). An average concentration of bile during the first 2 hours was 3 mcg/ml. 4. Irrespective of the route of administration (i.v., i.m., i.p. and s.c.), NTL was excreted rapidly in the urine and 70.0 approximately 81.0% of the dose was recovered within 6 hours. 5. Plasma level and urinary excretion of NTL after intramuscular administration (20 mg/kg) were almost similar to those of 5 other aminoglycoside antibiotics. 6. There was no difference between NTL and GM in plasma level after high dose (100 mg/kg, i.m).

[Absorption, distribution, metabolism and excretion of netilmicin in rats (III). Metabolic fate of 14C-netilmicin after intramuscular administration (author's transl)].

The metabolic fate of 14C-netilmicin (14C-NTL) was studied in rats after intramuscular administration (20 mg/kg). 1. Binding ratio of 14C-NTL to rat plasma protein, determined by the ultracentrifugal method, was 15 approximately 20% during the first 1 hour after intramuscular administration. 2. Binding ratio of 14C-NTL to HSA, determined by the equilibrium dialysis method, was 15 approximately 23%. Binding constant (K) and maximum binding number (n) were calculated as 1.48 X 10(4) and 3.3 mole/mole, respectively. Half of 14C-NTL bound to HSA was dissociated from HSA by gel-filtration. 3. The radioactivity in tail vein blood reached peak level at 10 minutes after intramuscular administration and declined rapidly. 4. Since the distribution rate of 14C-NTL into the blood cells was low during the first 2 hours, the plasma level at that time was about 1.5 times higher than whole blood level. 5. The recovery of radioactivity in the bile (0 approximately 24 hours) was only 0.13% of the dose administered. An average concentration during 2 hours after intramuscular injection was 2.5 mcg/ml. 6. Within 2 hours after administration, approximately 40% of the dose was recovered in the urine. Within 24 hours, 87.8% of the dose was excreted in the urine, 2.6% in the feces, 0.3% in the cage washing and 8.8% in the carcass. Total recovery ratio was 99.5% of the dose. 7. The radioactivity was widely distributed in the tissues, especially high in the kidney and bone, low in the brain. A high concentration of radioactivity was found in the renal cortex at 24 hours after administration. 8. Netilmicin was excreted into the urine unchanged and no metabolites were detected by TLC at all.

[Absorption, distribution, metabolism and excretion of netilmicin in rats (II). Accumulation study of netilmicin after repeated administration].

Repeated administration of netilmicin (NTL), a new aminoglycoside antibiotic, to rats at daily intramuscular dose of 10 or 20 mg/kg for 22 days did not affect the plasma level and the plasma half-life of the drug. The concentrations of NTL in the kidney increased markedly after repeated administration, and reached peak level after 8 and 15 doses for 10 and 20 mg/kg, respectively. Cumulative effect of NTL after repeated dose was also observed in the liver, spleen and lung, although the peak concentrations of NTL in these organs were below 1/50 of that in the kidney. Blood and tissue levels of NTL in rats were determined after a single intramuscular administration of 14C-NTL at a dose of 20 mg/kg following 21 repeated intramuscular administrations of NTL at daily dose of 20 mg/kg. The repeated dose of NTL had no effect on the blood level-time curve of radioactivity. The concentration of NTL in the kidney determined by radioassay was about 1/3 of that determined by bioassay, whereas the half-lives in the tissue levels determined by these two assays were nearly identical with each other. The half-life in the lung determined by bioassay was almost identical to that determined by radioassay, whereas the former was rather shorter than the latter in the liver and spleen.

[Absorption, distribution, metabolism and excretion of netilmicin in rats (IV). Distribution in kidney and transmigration to fetus or suckling (author's transl)].

Distribution in kidney and transmigration to fetus or suckling in rats were studied in male, pregnant or lactating rats after intramuscular administration of 14C-netilmicin (20 mg/kg). 1. After administration to male rats, the radioactivity in the kidney declined slowly with a half-life of approximately 6 days. 2. The radioactivity in the kidney was distributed in the renal cortex. The distribution pattern was further investigated by means of microautoradiography. The radioactivity was specifically observed in lysosomal granules of the proximal tubules at 6 hours after administration and reached maximum at 24 hours after administration, then declined gradually. On the other hand, the radioactivity in the distal tubules was lower than that of the proximal tubules. The highest radioactivity in the distal tubules was detected later than 24 hours after administration. 3. In pregnant rats (20th day of gestation), the distribution of radioactivity in the tissues were almost the same as those in the male rat. The small amount of radioactivity was detected in the fetal kidney and bone. 4. In mother rats (14 days after parturition), the radioactivity in the milk was 3 approximately 4 mcg equivalent of netilmicin/ml during 24 hours after administration. The small amount of radioactivity (0.13% of dose) was observed in the gastrointestinal contents of a suckling with 6 hours after administration.

[Subacute (5 weeks) subcutaneous toxicity study of cefsulodin using 3-week-old juvenile beagle dogs].

A subacute (5-week) subcutaneous toxicity study of cefsulodin (CFS) was carried out using 9 3-week old juvenile Beagle dogs. The dogs were distributed to 3 groups, each of which was constituted of 3 animals. Dogs in group I, II and III were given physiological saline (control), 300 mg/kg of cefazolin (CEZ, control drug) and 300 mg/kg of CFS, respectively. All animals used survived for 35 days of administration period. The changes, considered to be drug-related were histopathological changes at the sites of injection, which consisted inflammatory cellular infiltration and hyperplasia of fibroblast in subcutaneous tissue of skin. In terms of severity, CFS was less irritating than CEZ. CFS-related changes were not observed in other tests.

[Subacute toxicity study of cefotiam in three-week old beagle puppies (author's transl)].

A subacute (5-week) subcutaneous toxicity study of cefotiam (CTM) was carried out using 9 three-week old Beagle puppies. The puppies were assigned to one of three groups, each containing three. Puppies in group I (control) were given physiological saline; puppies in group II and III were given 300 mg/kg/day of CTM and cefazolin (CEZ), respectively. No behavioral abnormalities were seen in puppies in each group. The changes, considered to be drug-related, were histopathological changes at the sites of injection, which consisted inflammatory cellular infiltration, hemorrhage and hyperplasia of fibroblast in connective tissue of skin and skeletal muscle. In terms of severity, CTM was rather more irritating than CEZ. Except the histopathological changes described above, there observed no abnormalities which were considered to be related to CTM.