Mechanism of specific nuclear transport of adriamycin: the mode of nuclear translocation of adriamycin-proteasome complex.

Adriamycin (ADM), an anthracycline anticancer agent, is selectively stored in the nuclei of a variety of proliferating cells, but the precise mechanism of specific nuclear transport of ADM is not well known. Recently, we demonstrated that ADM shows high binding affinity to the cytoplasmic proteasomes of L1210 mouse leukemia cells and that taken up ADM by the cells selectively binds to proteasomes. Nuclear targeting of proteasome in proliferating cells may be mediated by the nuclear localization signals that are found in several of the alpha-type subunits of the 20S proteasome. To confirm nuclear transport of the ADM-proteasome complex, we synthesized a photoactive ADM analogue, N-(p-azidohenzoyl)-ADM, and generated a photoaffinity-labeled proteasome complex. The 26S proteasome purified from the cytosol of L1210 cells had a high affinity to N-(p-azidobenzoyl)-ADM. SDS-PAGE analysis of the photoaffinity-labeled proteasome showed that low molecular weight bands (approximately 21-31 kDa) of 20S proteasome had the highest photoaffinity. The photoaffinity-labeled proteasome was distributed in the cytoplasm and nuclei of digitonin-permeabilized L1210 and B-16 mouse melanoma cells in the presence of the cytosolic fraction and ATP. The rate of nuclear translocation of the proteasome was low in the absence of ATP. These results suggest that the proteasome is a specific translocator of ADM from the cytoplasm to the nucleus and that 20S proteasome components are the dominant ADM-binding sites. The nuclear transport of ADM-proteasome complex is regulated by an ATP-dependent nuclear pore-mediated mechanism.

Proprioceptive afferents survive in the masseter muscle of trkC knockout mice.

Peripheral innervation patterns of proprioceptive afferents from dorsal root ganglia and the mesencephalic trigeminal nucleus were assessed in trkC-deficient mice using immunohistochemistry for protein gene product 9.5 and parvalbumin. In trkC knockout mice, spinal proprioceptive afferents were completely absent in the limb skeletal muscles, M. biceps femoris and M. gastrocnemius, as previously reported. In these same animals, however, proprioceptive afferents from mesencephalic trigeminal nucleus innervated masseter muscles and formed primary endings of muscle spindles. Three wild-type mice averaged 35.7 spindle profiles (range: 31-41), six heterozygotes averaged 32.3 spindles (range: 27-41), and four homozygotes averaged 32.8 spindles (range: 26-42). Parvalbumin and Nissl staining of the brain stem showed approximately 50% surviving mesencephalic trigeminal sensory neurons in trkC-deficient mice. TrkC-/- mice (n = 5) had 309.4 +/- 15.9 mesencephalic trigeminal sensory cells versus 616.5 +/- 26.3 the sensory cells in trkC+/+ mice (n = 4). These data indicate that while mesencephalic trigeminal sensory neurons are significantly reduced in number by trkC deletion, they are not completely absent. Furthermore, unlike their spinal counterparts, trigeminal proprioceptive afferents survive and give rise to stretch receptor complexes in masseter muscles of trkC knockout mice. This indicates that spinal and mesencephalic trigeminal proprioceptive afferents have different neurotrophin-supporting system during survival and differentiation. It is likely that one or more other neurotrophin receptors expressed in mesencephalic trigeminal proprioceptive neurons of trkC knockout mice compensate for the lack of normal neurotrophin-3 signaling through trkC.

Differences in intracellular sites of action of Adriamycin in neoplastic and normal differentiated cells.

PURPOSE: This study was performed to clarify the intracellular specificity of the differential cytotoxic effects of Adriamycin (ADM) on neoplastic and normal cells. METHODS: The mouse lymphocytic leukemia cell line L1210 and pig kidney proximal tubular epithelial cell line LLC-PK1 were used as neoplastic and normal cells, respectively. These cells were treated with various concentrations of ADM for 24 h and toxicological parameters were determined. RESULTS: ADM (0.1-10 microM) significantly down-regulated cell growth rate and [3H]thymidine incorporation into DNA in the log phase, and at concentrations of more than 1 microM reduced the viability of both cell lines. Lipid peroxidation was increased at 1 microM ADM in L1210 cells and at 10 microM ADM in LLC-PK1 cells. The microsomal and nuclear fractions of both cell lines showed approximately the same level of ADM-induced superoxide anion (O2-) production, while the mitochondrial fraction of differentiated LLC-PK1 cells produced the highest levels of O2-. Differentiated LLC-PK1 cells showed the highest mitochondrial NADH-cytochrome c reductase activity. L1210 cells showed lower mitochondrial activities of enzymes involved in scavenging of reactive oxygen species, such as superoxide dismutase, glutathione peroxidase and catalase, than the other cells. CONCLUSIONS: These results suggest that ADM exerts cytostatic effects on neoplastic and normal undifferentiated cells through the inhibition of DNA synthesis by DNA intercalation, and cytotoxic effects on neoplastic cells through the accumulation of reactive oxygen species resulting from low scavenger enzyme activities. The cytotoxic effects on normal differentiated cells may be related to the high levels of production of reactive oxygen species due to high mitochondrial NADH-cytochrome c reductase activity.

Proteasome is a carrier to translocate doxorubicin from cytoplasm into nucleus.

When an effective concentration of doxorubicin (DXR) was added into L1210 of a mouse leukemia cell line, DXR was rapidly distributed much more in the nuclei than in the other organelle within a few minutes. A [14C]DXR-binding fraction was obtained from the cytosol prepared from L1210 cells. The fraction was adsorbed to hydroxylapatite matrix and eluted from the matrix by 50-150 mM potassium phosphate buffer. The fraction showed high DXR-binding and Suc-Leu-Leu-Val-Tyr-MCA-degrading activity. The binding of [14C]DXR was inhibited by unlabeled DXR. Gel chromatography of the fraction with Sephacryl S-300 separated two fractions of high molecular weight (Peak I, approx. 750 kDa) and low molecular weight (Peak II). Peak I showed proteolytic activity. [14C]DXR-binding Peak I had much higher affinity to DNA-cellulose than [14C]DXR-binding Peak II. [14C]DXR-Peak I complex also was retained into the nuclei isolated from L1210 cells, temperature-dependently. These results suggest that a specific carrier to translocate DXR from cytoplasm into nucleus exists in L1210 cell and the carrier is proteasome.

Micronucleus test with potassium chromate(VI) administered intraperitoneally and orally to mice.

The effect of route of administration, intraperitoneal (i.p.) or oral gavage (p.o.), in the mouse micronucleus test was studied with K2CrO4 in 2 mouse strains (MS/Ae and CD-1). A simplified acute toxicity test to estimate the toxic dose levels of K2CrO4 showed that the LD50S were 50 mg/kg i.p. and 300 mg/kg p.o. for MS/Ae and 32 mg/kg i.p. and 180 mg/kg p.o. for CD-1. Based on results of a pilot micronucleus test to determine appropriate dose levels and the optimal sampling time, it was decided to sample bone marrow cells of both strains of mice 24 h after i.p. doses of 10-80 mg/kg and p.o. doses ranging from 20 to 320 mg/kg. K2CrO4 administered i.p. induced micronucleated polychromatic erythrocytes (MNPCEs) dose-dependently in both strains. In contrast, when administered p.o. the chemical failed to induce MNPCEs. These results suggest that this difference between i.p. and p.o. routes is related to a difference of absorption or metabolic fate of chromate in vivo.

Dactimicin, a new, less toxic aminoglycoside antibiotic active against resistant bacteria.

Dactimicin is a new member of the pseudodisaccharide group of antibiotics. It possesses an unusual N-formimidoyl group which differentiates it from astromicin. Dactimicin is active against wide variety of bacteria, including resistant strains with aminoglycoside-modifying enzymes. However, AAC(3)-I enzyme slowly acetylates dactimicin. Animal toxicity studies show that the ototoxicity and nephrotoxicity of dactimicin are lower than those of amikacin and gentamicin. No notable abnormal findings have been found in pharmacological and toxicological studies.

Prevention by estradiol of aflatoxin-induced cytotoxicity in cultured chick embryo liver cells.

Aflatoxin B1 at a final concentration of 10(-5) M depressed the sytheses of DNA, RNA and protein in cultured chick embryo liver cells. The same dose produced ultrastructural changes in the nucleus, such as nucleolar compactness or segregation of fibrillar and granular components and in the cytoplasmic area, such as vesiculation, dilatation or degranulation of endoplasmic meticulum. These acute toxic effects of aflatoxin B1 were partially decreased by an addition of 4 X 10(-5) M estradiol-17 beta. Namely estradiol-17 beta significantly reduced the nucleolar compactness and segregation of fibrillo-granular components but did not improve the vesiculation, dilatation and degranulation of endoplasmic reticulum. Estradiol-17 beta also protected the liver cells from the aflatoxin B1-induced inhibition of nucleic acid and protein synthesis. These results suggest that the protective effect of estradiol-17 beta against the acute hepatotoxicity of aflatoxin B1 is mainly due to an antagonistic interaction of both compounds on the synthesis of nucleic acid in nucleolus.

Effect of aflatoxin B1 on the ultrastructural and biochemical development of chick embryo liver during organ culture.

When liver fragments from eleven-day chick embryos were maintained on Eagle's minimal essential medium by the established method of organ culture, they developed ultrastructural features similar to liver cells in vivo, except that they had small amounts of smooth endoplasmic reticulum and little glycogen. The cultured liver cells synthesized DNA, RNA and protein. The addition of aflatoxin B1 to the medium inhibited the synthesis of nucleic acid. Aflatoxin B1 also produced the segregation of granular and fibrillar components in nucleoli and the disarrangement of ribosomes attached to endoplasmic reticulum. Since these results were consistent with the known effects of the toxin in animals, we concluded that organ culture of chick embryo liver could be a useful technique for other studies.

Effect of repeated administrations of neomycin on the neuromuscular functions and the enhancement of d-tubocurarine action in mice.

Effects of repeated s.c. treatments with 50 mumol (approx. 31 mg)/kg/day of neomycin for 10 or 15 days were examined in mice. There were no significant differences between saline and neomycin treatment groups in the motor coordination assessed by rota-rod test and traction test. On tension recordings, an in vitro addition of d-tubocurarine inhibited the twitch tensions evoked by the nerve stimulations in both cases of saline or neomycin treatment. Neomycin treatments shifted the concentration-response curve between twitch tension and d-tubocurarine to the left, dependently on its injection days. These results suggest that repeated treatments with neomycin enhance the inhibitory effect of d-tubocurarine on the neuromuscular transmission in mice without eliciting motor incoordination and muscle relaxation.

Differential toxic effects of gentamicin on cultured renal epithelial cells (LLC-PK1) on application to the brush border membrane or the basolateral membrane.

Aminoglycoside antibiotics are generally accepted to accumulate in renal proximal tubule cells from the luminal surface and show toxic effects on the cells. The binding affinity and membrane permeability of aminoglycoside antibiotics are different at the brush border membrane (BBM) and the basolateral membrane (BLM) of proximal tubule cells. This study was performed, therefore, to investigate the differential effects of the aminoglycoside antibiotic gentamicin (GM) on cultured LLC-PK1 cells, a pig kidney proximal epithelial cell line, after addition to the BBM or the BLM side. LLC-PK1 cells were cultured on microporous membranes until forming confluent monolayers, and then GM was added to either the BBM or the BLM side. GM caused release of enzymes from the organelles, with a higher level of release observed following addition to the BBM side than that to the BLM side. Patterns of [3H]GM uptake by the cells differed in a manner dependent on whether it was added to the BBM or the BLM side. That is, the cellular uptake from the BBM side increased with incubation time, while that from the BLM side showed rapid saturation. These results suggested that aminoglycoside antibiotics show differential effects on cultured proximal epithelial cells and have differential patterns of cellular uptake when added to the BBM or the BLM side.

Roles of oxygen radical production and lipid peroxidation in the cytotoxicity of cephaloridine on cultured renal epithelial cells (LLC-PK1).

To clarify the mechanism of cephalosporin nephrotoxicity, the cytotoxic effects of cephaloridine (CER), a nephrotoxic cephalosporin antibiotic, on the pig kidney proximal tubular epithelial cell line (LLC-PK1) were studied in culture. CER increased the content of hydrogen peroxide and decreased the activity of catalase in the treated cells, followed by an increase in the content of lipid peroxide and decreases in both glutathione peroxidase activity and in the non-protein sulfhydryl content. The levels of NADPH-dependent hydrogen peroxide and superoxide anion production by microsomes prepared from LLC-PK1 cells, and by NADPH-cytochrome P-450 reductase purified from the rat renal cortex were significantly increased by paraquat. The production of these molecules was antagonized by p-chloromer-curibenzoate, an inhibitor of NADPH-cytochrome P-450 reductase. On the other hand, CER did not significantly affect the production of hydrogen peroxide or superoxide anions. These results suggested that the cytotoxic effect of CER on cultured LLC-PK1 cells was due to the increases in hydrogen peroxide and lipid peroxide levels and not microsomal oxygen radical production, and that the mechanism of this cytotoxicity is very different from that of paraquat which induces microsomal oxygen radical production.

Effect of aminoguanidine on chick embryonic liver during organ culture.

The addition of 10(-3) M aminoguanidine to culture medium caused no significant effect on the ultrastructure of cytoplasmic organellae in chick embryonic liver cells during organ culture but produced unique alterations in the nucleolar ultrastructure, such as increased density of protein matrix, increase of granular components, and fragmentation of nucleolar constituents. A high dose of aminoguanidine led the cultured liver cells to necrosis. Aminoguanidine also inhibited RNA synthesis rather than DNA synthesis in the cells. These results suggest that this agent may induce primary inhibition of RNA synthesis accompanied with significant changes of nucleolar structure in chick embryonic liver cells.

[Preventive effect of fosfomycin on the renal toxicity of cisplatin].

Cisplatin caused toxic effects in adult male rats, such as renal disturbance, decrease of platelet and WBC, increase of RBC, elevation of GPT and GOT activity, decrease of plasma protein and albumin, loss of body weight gain and lethal effect when treated intravenously with 1 mg/kg/day of cisplatin for 12 days. Fosfomycin (FOM) exerted preventive effects on the renal disturbance, the changes in blood cells and plasma protein and the lethal effect induced by cisplatin when treated with a combination of FOM and cisplatin. However, FOM did not prevent the cisplatin-induced effects on GPT and GOT activity and body weight gain. These results suggest that FOM prevents the cisplatin-induced disturbance of renal and hematopoietic function but does not the cisplatin-induced hepatic disturbance.

[Effect of fosfomycin on auditory organs and its transfer to cochlear lymph following application of its solution into a middle ear cavity].

Male guinea pigs were given 0.1 ml of 2, 3 or 5% fosfomycin (FOM) ototopical solution once a day for 5 days into a middle ear cavity through artificially perforated ear drum. Kanamycin A (KM) was used at 2% ototopical solution as control drug. Four animals of each group were sacrificed under pentobarbital anesthesia to isolate the cochlea 10 days after the final application. The cochlea was washed with 0.1 M phosphate buffer (pH 7.0), followed by fixing with 2.5% glutaraldehyde and 1% osmic acid. Cochlear specimens were prepared by standard method for scanning electron microscopic observation. The scanning electron microscopic observations revealed some damages in outer and inner hair cells, such as partial deformation or loss of auditory hair in hair cells, but these damages were not correlated to drug treatments. In order to determine the transfer of FOM and KM from middle ear cavity to cochlear lymph in male guinea pigs, the cochlear lymph was collected 0.5, 1, 2 and 4 hours after an application of 0.1 ml of 3 or 5% FOM and 2% KM ototopical solution into a middle ear cavity, followed by estimating content of these antibiotics in the lymph. The results showed the peak concentration in lymph at 1 to 2 hours after an application of 3% FOM was lower than that after 2% KM, but the AUC of 3% FOM was higher than that of 2% KM. The AUC value of FOM was dependent on the applied concentration of FOM. The value of half-life time was about 4.8 hours at FOM and about 2.3 hours at KM.(ABSTRACT TRUNCATED AT 250 WORDS)

[Toxicological studies on a new cephamycin, MT-141. VII. Its locally-irritating activity in rabbits].

The local irritation of MT-141 was compared with that of cefmetazole (CMZ) in rabbits to obtain following results. Microscopic observations revealed that the irritative activity of 10% solution of MT-141 in blood vessels was not so much different from that of saline and 10% solution of CMZ when they were injected twice a day into vein retroauricularis of rabbits for 7 days. The histopathological changes induced by 10% solution of MT-141 were similar to those by 10% solution of CMZ but somewhat different from those by saline, because both compounds caused slight necrosis in the tissue around vessels. Histopathological observations suggested that the occurrence of necrosis was due to the leakage of them during injections. The local irritation of MT-141 by an injection of 1 ml of its solution into muscle vastus lateralis was compared with that of CMZ in rabbits. The potencies of irritative activity of the test solutions were summarized in the following order; saline less than 5% MT-141 less than 10% MT-141 not equal to 10% CMZ much less than 0.75% acetic acid less than 6.0% acetic acid. The above-mentioned results suggest that MT-141 has low irritating activity when injected through intravenous or intramuscular route for clinical practice as 5% or 10% solution.

[Toxicological studies on a new cephamycin, MT-141. VIII. Its fertility test in rats].

A fertility study of MT-141 was performed in SD rats with the intramuscular (i.m.) injections at the dose levels of 400, 800 and 1,600 mg/kg/day. The male rats were injected with MT-141 for 63 days before mating and during the mating period, while the female rats were injected with MT-141 from the 14th day before mating up to the day 7 of gestation. All pregnant rats were sacrificed on day 20 of gestation followed by external, visceral and skeletal observations of their fetuses. The results are summarized as follows. The suppression of body weight gain was observed in males given above 800 mg/kg/day i.m. and in females of all treated groups during early period of gestation. However, no significant differences were found between treated groups and the control with regard to copulation rate and conception rate. Though no defects were observed for visceral and skeletal specimens in the fetuses of treated groups, MT-141 produced a delayed ossification of forelimbs in the fetuses at the doses above 800 mg/kg/day and of sternebrae at the dose of 1,600 mg/kg/day. It is concluded from the above-mentioned results that the maximal "no 'effective" dose of MT-141 on the fertility is above 1,600 mg/kg/day i.m. in parental rats and less than 800 mg/kg/day i.m. for the fetuses.

[Toxicological studies on a new cephamycin, MT-141. XI. Immunological properties].

Immunogenicity, eliciting antigenicity of MT-141 and its cross-reactivity with other beta-lactam antibiotics were studied in mice, guinea pigs and rabbits. The results were as follows. Injections of MT-141 failed to produce IgE-type antibody in mice but injections of the MT-141 conjugated to rabbit serum albumin produced a trace of IgE-type antibody. No antibody was produced in the guinea pigs immunized with the MT-141 conjugated to rabbit serum albumin in alum or Freund's complete adjuvant. The conjugated MT-141 also failed to elicit anaphylactic shock in the immunized guinea pigs. The subcutaneous treatments with MT-141 in Freund's complete adjuvant produced an amount of hemagglutination antibody in rabbits. The intravenous treatments with MT-141 produced no antibody in rabbits. When rabbits were subcutaneously immunized with the MT-141 conjugated to rabbit serum albumin in Freund's complete adjuvant, production of specific antibody in the rabbits was demonstrated by observations of passive cutaneous anaphylaxis and hemagglutination. The results of hapten-induced inhibition of passive hemagglutination, passive cutaneous anaphylaxis, anaphylactic shock and hemagglutination by using conjugates of antibiotics and rabbit serum albumin as immunogens and conjugates of antibiotics and bovine gamma-globulin as eliciting antigen showed that MT-141 did not cross-react with other antibiotics. MT-141 did not cause the in vitro direct Coombs' reaction in the human blood even at a high concentration of 160 mg/ml. It is concluded from these results that immunological activity of MT-141 preparation is weak.

[Toxicological studies on (2''R)-4'-O-tetrahydropyranyladriamycin, a new antitumor antibiotic. Its acute toxicity in rats].

(2''R)-4'-O-Tetrahydropyranyladriamycin (THP), a new antitumor antibiotic of anthracycline derivative, was given to Jcl-SD strain rats through intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c.) or oral (p.o.) administration routes and the animals were observed in respect of mortality, clinical signs and body weight for 21 days. Autopsy was done and histopathology on the tissues showing macroscopic abnormality was performed. The results were summarized as follows. Values of LD50 were 18.09 mg/kg i.v., 22.58 mg/kg i.p., 25.39 mg/kg s.c. and above 1,013 mg/kg p.o. for males and 18.07 mg/kg i.v., 20.30 mg/kg i.p., 21.76 mg/kg s.c. and above 1,013 mg/kg p.o. for females. No significant difference was found in LD50 values of different sexes. When higher than lethal dose levels of THP was given to animals, their clinical signs grew worse and weight loss occurred in about 5 days after the administration of the drug. Thereafter, deaths were observed. Macroscopic and microscopic observations on dead and survived rats revealed atrophy of spleen and thymus, whity clouding of spleen capsule, hemorrhage in mucosa of glandular stomach and congestion and hemorrhage in testes. These results suggest that THP shows weaker acute toxicity to rats than doxorubicin does, but the toxic effect of THP is approximately the same as that of other anthracycline derivatives.

[A study on the effect of (2"R)-4'-O-tetrahydropyranyladriamycin, a new antitumor antibiotic, on reproduction. I. Its effect on the fertility of rats].

The effect of a new antitumor antibiotic on the fertility was studied using SD rats. (2"R)-4'-O-Tetrahydropyranyladriamycin (THP) was administered to each rat at 0.01, 0.03 or 0.1 mg/kg daily. Males were given the drug intravenously for 63 days prior to mating and during the mating period; females were given the drug intravenously from 14 days prior to mating until day 7 of pregnancy. All the pregnant rats were sacrificed on day 20 of pregnancy, followed by external, visceral and skeletal observations of their fetuses. Results were summarized as follows. THP, at 0.1 mg/kg, suppressed body weight gain in females during the late period of pregnancy but did not affect body weight gain in males. THP, at 0.1 mg/kg, increased the numbers of dead fetuses and of resorptions. It caused no external, visceral or skeletal anomalies at any dose levels. The results suggest that, in rats, the maximum "no effect" dose of THP is 0.03 mg/kg/day intravenously regarding fertility and fetal development.

[A study on the effect of (2"R)-4'-O-tetrahydropyranyladriamycin, a new antitumor antibiotic, on reproduction. III. Its effects on perinatal and postnatal rats].

This paper describes effects of (2"R)-4'-O-tetrahydropyranyladriamycin hydrochloride (THP) on perinatal and postnatal rats. The drug was administered intravenously to female rats at 0.01, 0.03 or 0.1 mg/kg daily from day 17 of pregnancy to 21 days after delivery. Results were described below. At any dose levels tested, THP did not affect the body weight gain, food and water consumption by pregnant rats, and length of gestation period or delivery rate. However, at the highest dose level, THP decreased spleen weight. THP, at any dose levels, did not have toxic effect on development, physiological functions, behavior, mating, fertility or pregnancy of the first generation offspring (F1). At the highest dose of 0.1 mg/kg, however, THP produced delayed ossification of sacrococcygeal vertebra in the second generation fetuses (F2). The results suggest that the maximum "no effect" dose of THP to pregnant rats and offsprings is 0.03 mg/kg/day intravenously.