Mutagenic potential of alpha-(N2-deoxyguanosinyl)tamoxifen lesions, the major DNA adducts detected in endometrial tissues of patients treated with tamoxifen.

Breast cancer patients treated with the antiestrogen tamoxifen (TAM) show an increased risk of developing endometrial cancer. We have recently detected TAM-DNA adducts in endometrium obtained from patients treated with TAM and identified them as trans- and cis-forms of alpha-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM). To explore the mutagenic properties of these TAM-DNA adducts, we prepared site-specifically modified oligodeoxynucleotides containing a single isomer of dG-N2-TAM by reacting a 15-mer oligodeoxynucleotide containing a single dG (5'-TCCTCCTCGCCTCTC) with tamoxifen alpha-sulfate. These modified oligodeoxynucleotides were inserted into a single-stranded shuttle vector to investigate mutagenic specificities of the adducts in simian kidney (COS-7) cells. An epimer of dG-N2-trans-TAM showed targeted mutations ranging from 0.7 to 1.5%. The other dG-N2-trans-TAM adduct showed 9.6% G-->T transversions, accompanied by 2.8% G-->A transitions. Both dG-N2-cis-TAM adducts showed similar mutation spectra, where G-->T transversions (11-12%) predominated, along with a small number of G-->A transitions and G-->C transversions. Thus, dG-N2-TAMs are mutagenic lesions in mammalian cells. The tamoxifen-DNA adducts detected in patient endometrium may cause mutations and initiate endometrial cancer.

Alpha-hydroxytamoxifen is a substrate of hydroxysteroid (alcohol) sulfotransferase, resulting in tamoxifen DNA adducts.

When alpha-hydroxytamoxifen (alpha-OHTAM) was incubated with rat liver hydroxysteroid (alcohol) sulfotransferase a (STa) and 3'-phosphoadenosine 5'-phosphosulfate, (E)-alpha-OHTAM was found to be a better substrate for STa than (Z)-alpha-OHTAM. To explore the formation of tamoxifen (TAM)-derived DNA adducts, DNA was incubated with STa and either (E)-alpha-OHTAM or (Z)-alpha-OHTAM in the presence of 3'-phosphoadenosine 5'-phosphosulfate. Using 32P-postlabeling analysis, the amount of TAM-DNA adducts resulting from (E)-alpha-OHTAM was 29 times higher than that observed with (E)-alpha-OHTAM alone. Using (Z)-alpha-OHTAM and STa, some TAM-DNA adducts were also detected but at levels 6.5 times lower than that observed with (E)-alpha-OHTAM and STa. When compared with standards of stereoisomers of 2'-deoxyguanosine 3'-monophosphate-N2-tamoxifen, the major tamoxifen adduct was identified chromatographically as an epimer of the trans form of alpha-(N2-deoxyguanosinyl)tamoxifen, and the minor adduct was identified as an epimer of the cis form. In the reaction mixture, a conversion from (E)-alpha-OHTAM to (Z)-alpha-OHTAM through the carbocation intermediate was also detected. These results show that sulfation of alpha-OHTAM catalyzed by STa results in the formation of TAM-DNA adducts.

Mechanism of lower genotoxicity of toremifene compared with tamoxifen.

An increased incidence of endometrial cancer has been reported in breast cancer patients taking tamoxifen (TAM) and in healthy women participating in the TAM chemoprevention trials. Because TAM-DNA adducts are mutagenic and detected in the endometrium of women treated with TAM, TAM adducts are suspected to initiate the development of endometrial cancer. Treatment with TAM has been known to promote hepatocarcinoma in rats, but toremifene (TOR), a chlorinated TAM analogue, did not. TAM adducts are primarily formed via sulfonation of the alpha-hydroxylated TAM metabolites. To explore the mechanism of the lower genotoxicity of TOR, the formation of DNA adducts induced by TOR metabolites was measured using (32)P-postlabeling/ high-performance liquid chromatography analysis and compared with that of TAM metabolites. When alpha-hydroxytoremifene was incubated with DNA, 3'-phosphoadenosine 5'-phosphosulfate, and either rat or human hydroxysteroid sulfotransferase, the formation of DNA adducts was two orders of magnitude lower than that of alpha-hydroxytamoxifen. alpha-hydroxytoremifene was a poor substrate for rat and human hydroxysteroid sulfotransferases. In addition, the reactivity of alpha-acetoxytoremifene, a model activated form of TOR, with DNA was much lower than that of alpha-acetoxytamoxifen. Thus, TOR is likely to have lower genotoxicity than TAM. TOR may be a safer alternative by avoiding the development of endometrial cancer.

The Loss of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Caused by 24-Hour Rain Treatment Fully Explains the Decrease in the Photosynthetic Rate in Bean Leaves.

Recently, we have found that simulated rainfall causes a chronic inhibition of leaf photosynthesis in Phaseolus vulgaris (M. Ishibashi and I. Terashima [1995] Plant Cell Environ 18: 431-438). Mechanisms of this inhibition were examined in the present study. After the plants were treated with continuous mist for 24 h and then dried to unwet conditions, light-saturated photosynthetic rates of the leaves measured at 35 Pa ambient CO2 decreased to one-half of the control level. The extractable activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) also decreased to the same extent. Unexpectedly, this decline was due not to the lowered activation state but to the decrease in the amount of Rubisco. Before or after the "rain" treatment, the relationship between the net photosynthetic rate and the amount of Rubisco was expressed as a unique linear function with a small intercept (r2 = 0.84). From these it was inferred that the main cause of the rain-induced decline in photo-synthetic rate was the loss in amount of Rubisco.

Destruction of photosystem I iron-sulfur centers in leaves of Cucumis sativus L. by weak illumination at chilling temperatures.

The activity of photosystem (PS) I in cucumber leaves was selectively inhibited by weak illumination at chilling temperatures with almost no loss of P-700 content and PSII activity. The sites of inactivation in the reducing side of PSI were determined by EPR and flash photolysis. Measurement by EPR showed the destruction of iron-sulfur centers, FX, FA and FB, in parallel with the loss of quantum yield of electron transfer from diaminodurene to NADP+. Flash photolysis showed the increases in the triplet states of P-700 and antenna pigments, along with the decrease in the electron transfer from P-700 to FA/FB. This indicates the increase in the charge recombination between P-700+ and A0-. It is concluded that weak-light treatment of cucumber leaves at chilling temperature destroys FX, FA and FB and possibly A1. This gives the molecular basis for the mechanism of selective PSI photodamage that was recently reported [Sonoike and Terashima (1994) Planta 194, 287-293].

Inhibition of human O6-alkylguanine-DNA alkyltransferase and potentiation of the cytotoxicity of chloroethylnitrosourea by 4(6)-(benzyloxy)-2,6(4)-diamino-5-(nitro or nitroso)pyrimidine derivatives and analogues.

A series of 4(6)-(benzyloxy)-2,6(4)-diamino-5-(nitro or nitroso)pyrimidine derivatives and analogues of which 4(6)-benzyloxy groups were replaced with a (2-, 3-, or 4-fluorobenzyl)oxy or (2-, 3-, or 4-pyridylmethyl)oxy group, was synthesized. The abilities of these compounds to inhibit human O6-alkylguanine-DNA alkyltransferase (AGAT) in vitro and to potentiate the cytotoxicity of 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl) -3-nitrosourea (ACNU) toward HeLa S3 cells were evaluated. 2,4-Diamino-6-[(2-fluorobenzyl)oxy]-5-nitropyrimidine (3) and 2,4-diamino-5-nitro-6-(2-pyridylmethoxy)pyrimidine (6), whose ortho positions of the 6-substituent are modified, were much weaker in terms of these abilities than the corresponding meta- or para-modified compounds. These results are consistent with those of our previous study using a series of O6-benzylguanine derivatives. All 5-nitrosopyrimidine derivatives examined exerted both stronger AGAT-inhibition and ACNU-enhancement abilities than the corresponding 5-nitro derivatives. Among a variety of compounds that we have examined to date, 2,4-diamino-6-[(4-fluorobenzyl)oxy]-5-nitrosopyrimidine (10) exhibited the strongest ability to inhibit AGAT, and its magnitude was 2.5 and 50 times those of 4-(benzyloxy)-2,6-diamino-5-nitrosopyrimidine (9) and O6-benzylguanine (1), respectively. A strong positive correlation was observed between the ability to inhibit AGAT and to potentiate the cytotoxicity of ACNU. This strongly indicates that 4(6)-(benzyloxy)pyrimidine derivatives and their analogues potentiate ACNU cytotoxicity by inhibiting AGAT activity. To characterize the reactivity of test compounds, alkyl-transfer reactions were also carried out using the biomimetic alkyl-transfer system.

Enhancement effect of O6-fluorobenzylguanines on chloroethylnitrosourea cytotoxicity in tumor cells.

O6-Alkylguanine derivatives sensitize tumor cells to chloroethylnitrosourea (CENU) chemotherapy by inactivation of O6-methylguanine-DNA methyltransferase (MGMT), which repairs CENU-induced O6-alkylguanines in DNA by accepting the alkyl group at a cysteine moiety. To test the biological significance of synthesized O6-fluorobenzylguanine derivatives, we measured their ability of inactivation of MGMT activity and their effects on the cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in comparison with the effects of O6-benzylguanine and O6-phenylguanine. The O6-(4- and 3-fluorobenzyl)guanines considerably reduced the MGMT activity of HeLa S3 cell-free extract as did O6-benzylguanine. In contrast, O6-(2-fluorobenzyl)guanine and O6-phenylguanine had less of an effect on the activity. Two-hour pretreatment of O6-(4- and 3-fluorobenzyl) guanines potentiated ACNU cytotoxicity in HeLa S3 cells to a greater extent than did O6-(2-fluorobenzyl)guanine and O6-phenylguanine. The enhancement effects were consistent with the depletion of MGMT activity after the pretreatment of O6-fluorobenzylguanine derivatives. O6-Fluorobenzylguanines with a fluoro-substitution at the 4- or 3-position of the benzyl group were comparable to O6-benzylguanine and were powerful MGMT inactivators. The chemical features of the O6-benzyl group are a biologically important determinant in the reaction evolution with MGMT.

Acclimation of leaf characteristics of Fagus species to previous-year and current-year solar irradiances.

To examine the effects of different solar irradiances on leaf characteristics at the leaf primordium and expansion stages, we shaded parts of branches in the upper canopies of two adult beech trees, Fagus crenata Blume and Fagus japonica Maxim., for 4 years. The treatments during the leaf primordium and leaf expansion stages, respectively, were: (1) high light and high light (H, control), (2) high light and low light (HL), (3) low light and low light (LL), and (4) low light and high light (LH). Both number of cell layers in palisade tissue and individual leaf area were affected by the previous-year irradiance, whereas cell length of palisade tissue was larger in LH leaves than in LL leaves, suggesting determination by current-year irradiance. Lamina chlorophyll/nitrogen ratio was higher in HL and LL leaves than in LH leaves, suggesting determination by current-year irradiance. Diurnal minimum values of leaf water potential measured under sunlit conditions were lower in H and LH leaves than in HL and LL leaves. Effective osmotic adjustment was found in H and LH leaves, suggesting that leaf water relations were affected by current-year irradiance. Net photosynthetic rate and stomatal conductance measured under sunlight conditions were higher in H and LH leaves than in HL and LL leaves. Thus, effects of current-year irradiance had a greater effect on leaf-area-based daily carbon gain than previous-year irradiance.

The effect of internal CO2 conductance on leaf carbon isotope ratio.

We estimated internal CO2 conductance (gi) for C3 plant species of different life forms (annual herbs, deciduous trees and evergreen trees) grown in a variety of environments to examine the effect of gi on their leaf carbon isotope ratio (delta13C). The purpose of this study was to test the validity of using delta13C as an index of photosynthetic water use efficiency (WUE). When comparing deciduous tree species grown in contrasting light environments, there was a strong positive relationship between delta13C and WUE. Similarly, delta13C was positively correlated to WUE when comparing the different species of evergreen trees. However, the difference in WUE between evergreen and deciduous tree species did not relate to that in leaf delta13C. In addition, WUE was similar between highland and lowland herbaceous plants, although the former had a much higher delta13C. The positive relationship between delta13C and WUE did not hold across different life forms and different altitudes when differences in gi did not relate to those in stomatal conductance, resulting in independence of chloroplast CO2 partial pressure from intercellular CO2 partial pressure.

[Studies on 145 cases of septicemia in infancy and childhood--especially on septicemic patients with malignant and hematological diseases].

Clinical and bacteriological data of 145 inpatients with septicemia, treated at the hospital of Chiba University School of Medicine from 1972 to 1987, were reviewed by dividing them into three stages. (stages I: 1972-76, stage II: 1978-82, stage III: 1983-87) Patients with underlying diseases have been increasing: 91.8% of the total patients in stage III. Among the patients with underlying diseases, malignant and hematological diseases occupied about 60%, and in the other diseases, congenital heart diseases have been increasing in number. As to the organisms isolated, gram positive bacteria have increased, while gram negative bacteria have decreased. In stage III, the rate of gram positive organisms and gram negative ones accounted for 43.1% and 41.2% of all the isolates from the septic patients with malignant & hematological diseases, respectively. In patients with malignant & hematological diseases, alpha-streptococcus, coagulase-negative staphylococcus, and Fusobacterium sp. have been increasing, whereas, Escherichia coli, Enterobacter sp., Klebsiella sp., and Pseudomonas aeruginosa decreasing. In patients with other underlying diseases, S. aureus, CNS, and non Fermenters have been increasing. Among the patients without underlying diseases, gram positive bacteria accounted for the major part. The decrease of gram negative organisms in patients with malignant and hematological diseases may partially depend on the introduction of polymixin B as the drug of gut decontamination. The outcome of septicemia in the patients with malignant & hematological diseases has been markedly improving through all the three stages. During stage III, episode mortality and case mortality rate proved to be 23% and 31%, respectively. The introduction of the third generation cephems has decreased the mortality rate for gram negative organisms and contributed to the improvement of the total prognosis. The prognosis was worst in the case of P. aeruginosa, showing a mortality rate of 50% during stage III. Coincidence rate of blood and other cultures have been largest in the case of P. aeruginosa, so, the drug sensitivity of the strain cultured from other sites is sometimes useful in the choice of antibiotics.

[Influence of dexamethasone on the clinical course of bacterial meningitis in children. Especially on secondary fever. Experiences in 27 institutions].

Of pediatric patients with purulent meningitis seen at the institutions listed in the title page of this paper between 1986 and 1994, 93 patients treated with antibiotics and dexamethasone (DXM) were compared with 91 patients treated with antibiotics alone. The patients receiving antibiotics with dexamethasone achieved overall improvement in inflammatory symptoms and signs and cerebrospinal fluid findings and became afebrile significantly earlier than those receiving antibiotics alone. However, some of the patients became febrile again. The secondary fever rate for the DXM group was much higher than that for the antibiotic alone group (p < 0.0001). In most of the rebounded cases, the body temperature rose above 38 degrees C and remained elevated for 2-4 days. Cerebrospinal fluid (CSF) was cultured daily in 54 and 32 patients receiving antibiotics with and without DXM, respectively. Although this study was not a controlled study in a strict sense, these patients compared. In both groups, the CSF became mostly culture-negative within 48 hours. In a few patients receiving DXM, however, it became culture-negative after 72 hours or longer. DXM caused an adverse effect in a patient with meningitis caused by Streptococcus pneumoniae. The adverse effect was mild gastrointestinal bleeding, which recovered spontaneously. From the findings described above, the use of DXM combined with antibiotic therapy was considered to accelerate the relief from fever and improvement of inflammatory symptoms and signs and CSF findings. The body temperature rose again in more than half of the patients receiving DXM, but fell to normal spontaneously without treatment. The elevation doubtlessly could not be distinguished from recurrence of the meningitis itself or complications. It seems to be likely that no treatment but careful observation is required even if the fever recurs as far as the CSF findings showed favorable progress with excelluent general conditions. When DXM is given, it is essential that CSF tests and culture are repeated during the early stages and the progress is monitored carefully.

[Trend of bacterial meningitis in children over a 14 year period (1981 through 1994) in Japan--an analysis based on studies in 27 institutions].

We observed 266 children with purulent meningitis in 27 institutions in Japan during the 14 years from 1981 on dividing these years into 3 periods, 1981-1985, 1986-1990 and 1991-1994, and studied the trend of causative organisms identified in 254 among the 266 patients. Their ages were less than 3 months after birth in 50 children and 3 months or older in 216: there were 141 boys and 125 girls. The causative organisms were H. influenzae in 134 patients and S. pneumoniae in 50, most of them being aged 3 months or older. Next to the above bacteria ranked S. agalactiae in 29 and E. coli in 12, many of the patients were aged less than 3 months. Staphylococcus spp. was found in 7 patients and about 70% of them were aged 3 months or older. L. monocytogenes was found in 4 patients and N. meningitidis in 3 and they were aged 3 months or older in both patient groups. S. pyogenes, Enterococcus spp., Peptostreptococcus spp., P. Mirabilis and Enterobacter spp. were detected each in 1 patient. The causative organism was unknown in 21 patients and there was no double infection. H. influenzae were detected in 18 patients in 1981-1985 period (36.7%), in 56 in 1986-1990 (54.9%) and in 60 in 1991-1994 (63.8%) showing an increasing tendency, but S. pneumoniae exhibited neither an increasing nor decreasing tendency. There was a decreasing tendency with S. agalactiae and E. coli, but the details were not clear because there were few patients aged less than 3 months. Although the period of coexistence of 4 main bacterial species was not made clear in this study. Listeria is considered to develop mainly in the early childhood, and we believe that the conventional way of using a cephem preparation and ampicillin combined for patients under 6 years need not be altered. However, panipenem (phonetic) is likely to be effective for insensible S. pneumoniae for the time being.

[Guillain-Barré syndrome associated with anti-GQ1b antibody--nosological relationship between Fisher's syndrome and Guillain-Barré syndrome].

A 15-year-old man developed diplopia, ataxic gait and bulbar palsy. Two days after the onset of neurological symptoms, neurological examination revealed external ophthalmoplegia, cerebellar ataxia, and areflexia. Muscle weakness in the areas innervated by cranial nerves and in the four limbs, and glove and stocking type sensory impairment were also observed. On the 13th hospital day, CSF protein was elevated with normal cellularity. Serum IgM anti-GQ1b antibody was increased, which decreased concurrently with the clinical improvement. Recent studies have revealed the frequent presence of serum anti-GQ1b antibody in Fisher's syndrome. Therefore, this patient showed Fisher's syndrome at the beginning, then evolved to Guillain-Barré syndrome associated with anti-GQ1b antibody, which would support close association between Fisher's syndrome and Guillain-Barré syndrome.

[Clinical evaluation of a new macrolide antibiotic, azithromycin, in the pediatric field].

Azithromycin (AZM) preparations in fine granules and capsules were evaluated in 36 pediatric patients with various infections. In patients with pneumonia caused by Moraxella catarrhalis, Haemophilus influenzae or Mycoplasma pneumoniae, bronchitis, pharyngitis, scarlet fever, whooping cough, or campylobacter enteritis, AZM was found effective in 94.4% (34/36). As for the bacteriological efficacy of AZM, all of 12 strains identified were found eradicated by the treatment. Plasma T 1/2(24 approximately 48 hrs.) of AZM in fine granules, given two patients at 10 mg/kg body weight once daily for 3 days, were 41.5 and 51.4 hours, while AUC0 approximately infinity was 7.45 and 13.44 mg.hr/ml. The rates of AZM recovered in the urine samples from two pediatrics patients in the first 81 hours of treatment, when it is given in fine granules at 10 mg/kg body weight once daily for 3 days, were 6.27% and 11.0%. Data from 43 patients were included for drug safety evaluation. Neither adverse reactions nor abnormal laboratory changes were observed. In conclusion, AZM was found useful in treatment of pediatric infections.

[Clinical evaluation of cefozopran in the pediatric field].

A new parenteral cephem, cefozopran (CZOP), was evaluated for its safety and efficacy in 21 children with acute infections. A mild side effect or an abnormality in laboratory tests was observed in one case each, but the safety of CZOP was otherwise observed. CZOP was effective in all of the 18 bacterial infections tested including pneumonia, cellulitis and urinary tract infections, and all the causative organisms were eradicated. Serum half-lives of CZOP were 1.5-1.8 hours and the urinary excretion rates were 63-96% in the first 5-8 hours after administration. These data suggest that CZOP is safe and effective in children with susceptible bacterial infections.

[Clinical evaluation of a new oral penem, SY5555, in the pediatric field].

A new oral penem antibiotic, SY5555, was evaluated for its safety and efficacy in 35 children with various bacterial infections. SY5555 was effective in 100% of scarlet fever, pharyngotonsillitis, pneumonia, otitis media, bacterial diarrhea, urinary tract infections and skin and soft tissue infections. The etiologic bacteria were eradicated except Salmonella sp. Side effects were observed in 3.5% cases; one was diarrhea and Candida dermatitis, one was loose stool, and one was Candida dermatitis. From these data, SY5555 is thought to be a safe and effective antibiotic in the pediatric field. Regular dose of suspension preparation is 15 mg/kg/day in 3 divided dosages, and when needed the dose may be doubled.

[Clinical evaluation of S-1108 in children].

A new oral cephem antibiotic, S-1108, was evaluated for its clinical efficacy and safety in children. S-1108 was effective in 95% of the 59 examined cases of respiratory, middle ear, skin and urinary tract infections. S-1108 was highly effective in infections of Streptococcus pyogenes, Haemophilus influenzae and Escherichia coli, but was less effective in penicillin-resistant Streptococcus pneumoniae and Staphylococcus aureus infections. The serum half-life was 1.26 +/- 0.36 hours upon after meal administration of 4 mg/kg. No severe adverse reaction was encountered. From these data, S-1108 appears to be safe and effective in children with susceptible bacterial infections.

[A clinical study on cefditoren pivoxil granules in the pediatric field].

The clinical efficacy of cefditoren pivoxil (CDTR-PI, ME 1207) was evaluated in 45 patients with various infections. CDTR-PI was administered after meals at a dose of 3 mg/kg t.i.d. to most patients. The clinical efficacy rate was 95.3%. As side effects, diarrhea occurred in 2 patients. Cefditoren showed excellent antibacterial activities against Haemophilus influenzae, Streptococcus pyogenes and Streptococcus pneumoniae, and was more effective against Staphylococcus aureus than other cephems.

[Clinical evaluation of cefdinir 10% granules in children].

Cefdinir (CFDN, FK482) was evaluated in children with infections. CFDN was given at a daily dose of 6.4-19.8 mg/kg in 2 or 3 divided portions. CFDN was effective in 94% of 32 cases with respiratory tract, middle ear, urinary tract or skin structure infections. Side effects were loose stool and diarrhea (12.5%). In a pharmacokinetic study, 6.0 mg/kg of CFDN was given to each of the subjects before meal. Cmax was 0.81 +/- 0.38 microgram/ml, T 1/2 was 2.31 +/- 0.77 hours. Antibacterial activity against Staphylococcus aureus was the most excellent of oral cephem antibiotics tested. The data suggest that CFDN 10% granular preparation is safe and effective when used in children with infections caused by susceptible bacteria.

[Pharmacokinetic, bacteriological and clinical studies on cefozopran in neonates].

The efficacy, safety and pharmacokinetics of cefozopran (CZOP) were evaluated in neonates and the following results were obtained: 1. Of the 12 patients treated with CZOP, judgment of clinical efficacy was evaluable in 10 patients (including 5 with pneumonia and 3 with urinary tract infections). The treatment was effective and the causative organism was eradicated in 100% of the patients. 2. No adverse signs and symptoms were recognized during the treatment with CZOP. A slight elevation of direct bilirubin was recognized as an abnormal alteration of laboratory test values in one patient. The value, however, returned to the normal range after the completion of treatment. 3. The pharmacokinetic evaluation was made in 3 of the 12 patients. The blood CZOP levels were recognized in proportion with the dosages. The elimination half lives (T 1/2) in those patients were 8.92, 2.90 and 2.76 hours. Prolongation of T 1/2 was recognized in the patient aged 0 day. It was possible to examine the urinary excretion only in one patient aged 18 days. The excretion rate of the drug was 68.6% of dose by 8 hours after administration. These results suggest that CZOP is a drug useful for treatment of infections in neonates as well, with high efficacy and safety.