Effects of probenecid on the pharmacokinetics of mizoribine and co-administration of the two drugs in patients with nephrotic syndrome.

Probenecid (PRB) is an agent that reduces the systemic level of uric acid, and has the ability to inhibit the renal tubular secretion of agents that are co-administered with it. In this study, we evaluated the effects of PRB co-administered with mizoribine (MZR) on the pharmacokinetics (PK) of MZR in 12 patients with nephrotic syndrome. The elimination rate constant (kel) was used as an indicator of changes in the PK of MZR when the secretion of MZR was inhibited by co-administration of PRB, in order to determine the extent to which MZR was influenced by PRB. In 4 of the 12 patients studied, kel decreased and the biological half-life (t1/2) of MZR was prolonged when co-administered with PRB, in comparison with the values when MZR was used alone, thus revealing that the PK of MZR was influenced by PRB. Co-administration of PRB with MZR appears to be effective in prolonging the biological half-life of MZR and enhancing its effect in patients with nephrotic syndrome, although further studies will be required to determine the optimal dosage of PRB and renoprotective effects.

Sodium-sensitive variability of the antiproteinuric efficacy of RAS inhibitors in outpatients with IgA nephropathy.

AIMS: Inhibition of the renin-angiotensin system (RAS) decreases proteinuria in IgA nephropathy and often retards disease progression. However, its antiproteinuric efficacy varies considerably among patients or different stages in a single patient. We sought for the factor(s) underlying the variation in urinary protein excretion in RAS inhibitor-treated outpatients with IgA nephropathy. PATIENTS: 43 patients with biopsy-proven IgA nephropathy, moderate proteinuria (0.5 - 3.5 g/day), normal to moderately-low estimated GFR (eGFR) (28.6 - 114.2 ml/min/1.73 m2) and normal blood pressure, prehypertension or mild hypertension (systolic/diastolic blood pressures < 160/100 mmHg) were placed on RAS inhibitors following diagnosis. METHOD: Excretion of urinary protein (UprV) and sodium (UNaV), estimated protein intake (EPI) and the mean blood pressure (MBP) were determined on 12 consecutive visits for an average duration of 17.6 months. Analyses were performed to determine which factor(s) influenced the variation in UprV. RESULTS: 14 patients (32.6%) showed a significant correlation between UprV and UNaV, whereas UprV correlated significantly with EPI or MBP in 7 (16.3%) and 3 patients (7.0%), respectively. The 14 patients were characterized by lower eGFR and more extensive glomerulosclerosis and tubulointerstitial damage at baseline than the other 29 patients. The UprV-UNaV correlation was significant in 8 of 12 patients (66.7%) with eGFR < 60 ml/min/1.73 m2 and in 6 of 29 patients (19.4%) with eGFR >= 60 ml/min/1.73 m2 (p < 0.05). The UprV/UNaV regression lines were significantly steeper with more extensive glomerulosclerosis (p < 0.05) and tubulointerstitial damage (p < 0.05) at baseline. The lines also tended to be steeper with lower baseline eGFR (p = 0.062). CONCLUSIONS: These results showed that the antiproteinuric effect of RAS inhibitors becomes susceptible to an increase in urinary sodium excretion as renal function and functioning nephron mass decline with the progression of renal histological damage. Stringent dietary sodium restriction is required to maximize the antiproteinuric effect of RAS inhibitors in outpatients with IgA nephropathy.

Urinary type IV collagen excretion reflects renal morphological alterations and type IV collagen expression in patients with type 2 diabetes mellitus.

AIM: We tried to establish the significance of quantifying urinary type IV collagen (IV-C) excretion for the evaluation of renal involvement of type 2-diabetic patients. METHODS: Twenty patients (13 males and 7 females; age range 31 to 69 years) with type 2 diabetes mellitus had undergone renal biopsy and relationship between the severity of morphological alteration, IV-C expression and urinary IV-C excretion were examined. RESULTS: Urinary IV-C excretion significantly correlated with mesangial expansion score (p = 0.49, p < 0.05) and tubulointerstitial injury score (p = 0.56, p < 0.05). Furthermore, urinary IV-C excretion significantly correlated with both glomerular (r = 0.56, p < 0.01) and tubulointerstitial IV-C expression areas. Urinary protein excretion also correlated with mesangial expansion score and tubulointerstitial injury score. However, it did not correlate with the expression of IV-C in the kidney. CONCLUSIONS: These results suggest that urinary IV-C excretion reflects the pathogenetic process of diabetic nephropathy, which urinary protein excretion alone cannot do sufficiently. It can be concluded that urinary IV-C excretion could be a more useful marker for the evaluation of renal involvement of type 2-diabetic patients.

Evaluation of Mycoplasma hyopneumoniae inactivated vaccine in pigs under field conditions.

An inactivated vaccine prepared from broth culture supernatant of Mycoplasma hyopneumoniae with an aluminum adjuvant was evaluated in three herds (herd A: specific pathogen-free herd, herd B: high health status herd with no clinical signs of respiratory infection, herd C: low health status herd with serious epidemiological and economical problems). A total of 212 pigs from the three herds were divided into two groups. One group was injected twice with the vaccine at 4-week intervals and the other was a control group. No adverse reactions were noted following the vaccinations either systematically or locally in any of the vaccinated pigs from any of the herds. In herd A, the vaccination provided antibody response within 4 weeks after the second vaccination and antibody responses continued for more than 12 weeks. In herds B and C, the number of pigs with lung lesions, mean percentage of lung lesions, and the numbers of M. hyopneumoniae recovered from pigs at slaughter in the vaccinated group were significantly (P < 0.05) reduced compared to the control group. Furthermore, vaccination resulted in improved average daily weight gain (ADG), improved feed conversion ratio (FCR), and improved days to market weight in herd C, whereas no difference in growth performance was shown in herd B. It is suggested that the inactivated vaccine prepared from broth culture supernatant of M. hyopneumoniae is effective in reducing clinical signs and lung lesions. Also, vaccination resulted in improved growth performance in herds where clinical signs and economic losses were significant.

Agreement of mutational characteristics of heterocyclic amines in lacI of the Big Blue mouse with those in tumor related genes in rodents.

The mutational spectra of carcinogenic heterocyclic amines (HCAs), 2-amino-3,4-dimethylimidazo[4,5-b]quinoline (MeIQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-9H-pyrido[2,3-b]indole (A alphaC) were studied in the colon of Big Blue mice. In 90, 115 and 105 lacI mutants from mice fed 300 p.p.m. MeIQ, 400 p.p.m. PhIP and 800 p.p.m. A alphaC, respectively, 92, 115 and 105 mutations were identified. G:C-->T:A transversions predominated with these HCAs. Mutational hot spots for base-substitution mutations caused by MeIQ, PhIP and A alphaC were in distinct sequence contexts; at 5'-GC-3', in runs of guanine and in 5'-CGT-3', respectively. Further, 30 of 115 (26%) PhIP-induced mutations were G:C base pair deletions, and eight of these deletions were in 5'-GGGA-3'. The mutational characteristics of MeIQ in the lacI gene coincided well with those in the Ha-ras gene of MeIQ-induced mouse forestomach tumors and rat Zymbal gland tumors. The characteristic single-base deletion induced by PhIP in the lacI gene also coincided well with those in the Apc gene of PhIP-induced rat colon tumors. These results suggest that the mutational characteristics of each chemical are conserved across different genes in different species.

BRCA2 germline mutations in Japanese breast cancer families.

Germline mutations of BRCA2 were examined in 20 Japanese breast cancer families without BRCA1 mutations, including one demonstrating cancer development in a male. Three different mutations, resulting in truncation of the BRCA2 protein, were detected in 3 different families. They were 9474insA (exon 24, termination at codon 3110), C8729A (exon 20, S2834 ter) and 982del4 (exon 9, termination at codon 275). The 982del4 mutation was detected in the family with a case of male breast cancer. Age at onset was young, with a range of 28-43 years, in the 2 female breast cancer families with truncation mutations. One probable missense mutation, A10462G (13412V), was further detected in 2 families, although cosegregation of this allele with the breast cancer phenotype was not complete. The rate of BRCA2 mutations in Japanese families was suggested to be almost the same as in Western countries, and larger than it is the case for BRCA1.

Establishment of methylation-sensitive-representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors.

Methylation of CpG sites in the genome, which is generally conserved during cell replication, is considered to play important roles in cell differentiation and carcinogenesis. However, investigations on changes in methylation status have been limited to known genes. To make a genome-wide search for differentially methylated genes, we developed a methylation-sensitive-representational difference analysis (MS-RDA) method. The representation of the genome was prepared using the methylation-sensitive restriction enzyme HpaII, and the mixture ratio of tester and driver DNAs was optimized to detect differences in methylation status of a single copy per diploid mammalian genome. By performing comparative MS-RDA of one hepatocellular carcinoma and of background liver tissue of one mouse treated with a food carcinogen (2-amino-3,4-dimethylimidazo[4,5-f] quinoline), we were able to identify (i) extensive hypomethylation of long interspersed nuclear element repetitive sequences in a number of hepatocellular carcinomas, (ii) reduction of the gene dosage of their mitochondrial DNA, and (iii) a hypermethylated DNA fragment of unknown origin. Furthermore, by adding the clones obtained in the first MS-RDA to the driver DNA [MS-RDA with elimination of excessive clones (MS-RDA-WEEC)], nine DNA fragments that could not be detected at the first MS-RDA were isolated as differentially methylated DNA fragments. MS-RDA, combined with MS-RDA-WEEC, is thus a promising approach to identify DNA fragments differentially methylated in two DNA sources.

Induction of aberrant crypt foci in C57BL/6N mice by 2-amino-9H-pyrido[2,3-b]indole (A alphaC) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx).

The inductions of aberrant crypt foci (ACF) by two carcinogenic heterocyclic amines (HCAs), 2-amino-9H-pyrido[2,3-b]indole (A alphaC) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), were studied in the large intestine of C57BL/6N mice. Seven-week-old mice were fed a diet supplemented with 500 or 800 ppm of A alphaC, or 400 or 600 ppm of MeIQx for 7 weeks, followed by a basal diet for another 7 weeks. A alphaC at 800 ppm induced 8.0 +/- 1.9 and 7.8 +/- 2.5 ACF in female and male mice, respectively. MeIQx at 600 ppm induced 2.8 +/- 1.8 and 1.6 +/- 0.8 ACF in females and males, respectively. At lower concentrations of A alphaC and MeIQx, many fewer ACF were induced. No ACF were induced in the control group. The size of ACF (number of aberrant crypts/ACF) in all experimental groups was between 1.0 and 1.5. More than half the A alphaC-induced ACF were located in the region about 20-40% of the distance from the ileocecal portion to the anus. Although both of these HCAs were reported to induce tumors in the liver and other organs, but not in the large intestine, of CDF1 mice, these findings suggest that both these HCAs, and especially A alphaC, induce large intestinal tumors in C57BL/6N mice.

Effects of gender and species on spectra of mutation induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the lacI transgene.

Feeding of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to F344 rats induces colon tumors specifically in male rats. Mutant frequencies and mutational spectra of the lacI transgene were studied in male and female Big Blue transgenic rats after feeding 400 ppm of PhIP in the diet for 60 days. Mutant frequencies in the colon mucosa were increased 20-25 times compared with those of the control rats, being 661.4 +/- 33.3 x 10(-6) and 718.2 +/- 16.9 x 10(-6) in males and females, respectively. No significant differences in types and distribution of the mutations were detected between males and females. One-base deletions were the most frequent mutation, including the characteristic guanine deletion at 5'-GGGA-3' which is also seen in the Apc gene of rat colon cancers induced by PhIP. Comparison of the lacI mutations in the rat colon with those previously identified in the mouse colon showed that the rate of G to T transversions was significantly higher in the mouse. This is the first report stating that there exist differences in the mutation specificity on the same gene, among mammalian species. However, the characteristic guanine deletion was recovered in both the mouse and the rat. These findings do not offer a mechanistic explanation of the gender specificity of PhIP-induced colon cancer in rats, though the universality of the guanine deletion suggests that this alteration may prove a useful indicator of human exposure.

The role of an uncommon type of oral streptococcus sanguis in the etiology of behcet's disease.

The relationships of Behcet's disease (BD) with oral diseases and the prevalence of an uncommon type of oralStreptococcus sanguis (Str. sanguis) in the oral cavity were investigated in a case-control study. BD patients were compared to patient controls (collagen disease) and healthy controls.An interview questionnaire survey of BD and oral diseases showed that during the pre-onset, onset, and post-onset periods, the incidences of tonsillitis and dental caries, or the history of dental treatment, were greater in BD cases. Typological analysis showed a higher prevalence of an uncommon type ofStr. sanguis, differing from the common type, among BD cases compared to control groups. These results, showing a higher incidence of tonsillitis and dental caries during the presymptomatic period, a greater frequency of dental treatments during the symptomatic period, and the presence of an uncommon type ofStr. sanguis, indicate thatStr. sanguis of an uncommon type is related to increased risk of BD, and the possibility of a causal role is suggested.

The effects of a 4.7 tesla static magnetic field on the frequency of micronucleated cells induced by mitomycin C.

The effects of a 4.7 tesla (T) static magnetic field (SMF) on the frequency of micronucleated cells (MN-cells) in CHL/IU cells induced by mitomycin C (MMC) were studied in vitro. Exposure to a 4.7 T SMF for 6 hr significantly decreased the frequency of MMC-induced MN-cells for expression culture periods of 18, 42, 54 and 66 hr. The highest frequency of MMC-induced MN-cells was observed at the expression culture period of 42 hr and decreased gradually in the same manner in both exposed and control groups. These results suggest that a 4.7 T SMF may exert on influence during the DNA damage stage produced by MMC rather than on the formation of micronuclei during the stage following MMC-induced DNA damage.

Metallothionein content increased in the liver of mice exposed to magnetic fields.

Although recent studies have shown that various stress can induce metallothionein (MT) synthesis in animal tissues, the induction of MT synthesis by exposure to static magnetic fields (SMF) has not been reported. We measured MT levels in the liver, kidney and brain of mice exposed to SMF and also evaluated the effect of SMF exposure on the induction of hepatic MT by treatment with carbon tetrachloride (CCl4). The MT content in the liver was significantly increased by exposure to 4.7 T of SMF for 6, 24, or 48 h, whereas that in the kidney or brain was not changed compared to the control. The combination of CCl4 injection and SMF exposure induced elevation of the hepatic MT content exceeding that induced by either treatment alone. These results indicate that exposure to the strong SMF induces MT synthesis in the liver in mice and enhances the hepatic MT synthesis induced by CCl4 administration.

[Biological effects of electromagnetic fields].

Since several epidemiological studies have indicated an elevated risk for certain types of cancer in both living and working environments where exposure to an extremely low-frequency electromagnetic field (ELF) occurs, public concern about ELF has been increasing because it is impossible to imagine life today without electricity. We reviewed studies on biological effects of ELF with respect to their cytological and biochemical effects, including mutagenicity, clastogenicity and carcinogenicity. The studies can be summarized as follow: 1) There is evidence that outer surface of the cell membrane is the primary locus for ELF-induced cellular alterations. 2) ELF modulate the proliferation of normal as well as transformed cells in vivo and in vitro. The magnitude of the proliferative effects depends on ELF intensity, exposure duration and other cellular factors. 3) No studies clearly demonstrate deleterious effects of ELF exposure on mammalian reproduction and development, but several suggest such effects. 4) Reported evidence does not demonstrate that the ELF acts as a cancer initiator. However, it might act as a promoter or affect tumor progression. Further observations and epidemiological studies of ELF must be accompanied by laboratory experiments to evaluate biological and health effects.

Mutation, loss of heterozygosity, and recombination of the p53 gene in mouse forestomach tumors induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline.

2-Amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), a food mutagen, induces forestomach tumors in CDF1 mice. We established a polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis system to detect mutations in the mouse p53 gene exons 2-10, which encompass all five regions conserved among species, and a system to examine loss of heterozygosity (LOH) that uses newly identified polymorphisms between BALB/c and DBA mice, the parental strains of CDF1 mice. Four original forestomach tumors (one papilloma, two carcinomas, and one lymph-node metastasis) and four cell lines derived from four independent forestomach tumors were examined with the PCR-SSCP system and by polymorphism analysis. Of the four original tumors, the papilloma had a G-->A transition at the second position of codon 171, and one carcinoma had a G-->T transversion at the second position of codon 113 with loss of the wild-type allele, whereas the other two carcinomas had no detectable mutations. Of the four cell lines, two had a base substitution and LOH, and the other two had double mutations (a base substitution and a deletion). By amplification of the double mutations in a fragment, the two cell lines were shown to have four kinds of alleles, indicating induction of recombination within the p53 gene. Our results show that our PCR-SSCP analysis system is efficient for detecting p53 mutations in mouse genomic DNA and that alteration of the p53 gene plays a significant role in MeIQ-induced mouse forestomach carcinogenesis.

Effects of prostaglandin E2 on the micronucleus formation in mouse bone marrow cells by various mutagens.

The effects of prostaglandin E2 (PGE2), as a trigger of erythroid progenitor cells into the cell cycle, were studied on the induction of micronuclei by various mutagens; with mitomicin C (MMC) the optimal protocol was established. PGE2 itself did not induce any micronuclei in this experiment. The highest frequency of micronuclei and dose-response relationship between PGE2 doses and micronucleus frequency were observed 30 h after injection of MMC to mice administered PGE2 24 h previously. Sensitization by PGE2 pretreatment was also found for other mutagens, such as vincristine, 5-fluorouracil, benzo[a]pyrene, 1,1-dimethylhydrazine and 2-naphthylamine. These results support the hypothesis that accelerating the erythropoiesis increases the frequency of micronuclei induced by mutagens.

[A basic study for estimating the level of exposure to a nasal inhalant containing a stimulant].

Criminal cases involving stimulant abuse have increased since 1970 but have now leveled off. Some of the offenders claimed to have used the Vicks Inhaler containing a stimulant (1-methamphetamine) which is used for the treatment of nasal obstruction. The aim of this experiment was to measure the amount of 1-methamphetamine contained in the Vicks Inhaler by stimulating the human respiratory system. The results are as follows: 1) The data from the stimulation experiment showed that the inhalation level of 1-methamphetamine was estimated to be 320.4ng. From this value, the level of 1-methamphetamine absorbed per one respiration was calculated to be 21ng. 2) The data from quantitative and qualitative analysis by gas-chromatography showed that menthol interfered with the methamphetamine. 3) A qualitative test for the stimulant in urine was negative when the subject inhaled the Vicks Inhaler only once. However, this test turned positive when the subject inhaled it more than 17 times.

Micronucleus test and erythropoiesis: effect of cobalt on the induction of micronuclei by mutagens.

The micronucleus test is used widely as an in vivo short-term assay for potential carcinogens. In the present study, results of the micronucleus test were affected by cobalt dichloride pretreatment. Cobalt dichloride was used to induce erythropoietin, a growth factor for erythropoiesis. The increase in mutagen-induced micronucleus response following cobalt pretreatment, therefore, may have been due to a change in the rate of erythropoiesis. The greatest interaction between cobalt pretreatment and mutagen treatment for the induction of micronucleated polychromatic erythrocytes (MPCE) occurred when mice were injected with 1,1-dimethylhydrazine (DMH) 12-24 hr after pretreatment with cobalt dichloride and killed 30 hr later. Increased sensitivity of the micronucleus test was attributable to the administration of mutagen during the differentiation and multiplication of erythroblast, which is presumed to have been accelerated by pretreatment with cobalt dichloride. An increased induction of MPCE in the bone marrow by two chemicals--benzo(a)pyrene, 2-naphthylamine--was also observed following pretreatment with cobalt dichloride.

[A case of Kawasaki's disease combined with septicemia--isolation of Streptococcus sanguis (MCLS-1) and Streptococcus pyogenes from blood at the acute stage].

The patient was diagnosed as having Kawasaki's disease. Streptococci were isolated from cultures of all blood samples collected during the acute stage (the third, fifth and seventh day of the disease). The streptococci were subsequently identified as Streptococcus sanguis (MCLS-1) and Streptococcus pyogenes. This finding may suggest induction of Kawasaki's disease by S. sanguis MCLS-1, and in this case, the complication of septicemia by S. pyogenes.