Study on Differences of Inducible Nitric Oxide Synthase Gene Expression in Vesical Tissue after Intravesical Instillation of Three Different strains of BCG in Rat / 대한비뇨기과학회지

Recently, BCG infection is known to induce nitric oxide(NO) production by macrophages through T cell mediated process and NO is known to be microbicidal and tumoricidal. There are several strains of BCG which are commercially available and vary in the number, pathogenicity, viability, and immunogenicity of organisms. Therefore, we wanted to know if there are any differences between three different strains of BCG(Pasteur, Connaught or Tice strain) on the induction of inducible NO synthase(iNOS) and the histological changes in vesical tissue after intravesical instillation of BCG in rats. Thirty two Sprauge-Dowley female rats were equally divided into 4 groups. In group 1, normal saline(0.85 ml/kg) was intravesically instilled one time. In group 2, 3, and 4, BCG of Pasteur strain(2mg/kg, normal saline 0.85ml/kg), Connaught strain(1.35mg/kg, normal saline 0.85ml/kg), Tice strain(0.21mg/kg, normal saline 0.85ml/kg) was instilled one time, respectively. The bladders were excised from each group on day 1, 3, 7, and 14 after BCG instillation. iNOS mRNA was not detected in the vesical tissues of control group, whereas it was strongly detected in group 2, 3, or 4. Also, iNOS mRNA was more strongly detected on day 1, 3, and 7 after intravesical BCG instillation than day 14 in the vesical tissues of group 2, 3, and 4. Histologic findings were well related with expression of iNOS mRNA. Our results indicate that intravesical BCG instillation of rat induces expression of iNOS mRNA in the vesical tissue accompanying the infiltration of inflammatory cells and suggest that all of the 3 strains of BCG including Pasteur, Connaught, and Tice are good at inducing expression of iNOS mRNA without significant differences between the strains.

Expression of Inducible Nitric Oxide Synthase in Vesical Tissue after Intravesical BCG Instillation in Rat / 대한비뇨기과학회지

Intravesical bacillus calmette-guerin( BCG) therapy for superficial bladder carcinoma is believed to exert its antitumor effects through immune mechanisms which have yet to be more clearly defined. Recently, BCG infection is known to induce nitric oxide(NO) production by macrophages through a T cell mediated process. NO is known to be microbicidal and tumoricidal. Therefore, we studied the effects of intravesical BCG instillation on the induction of inducible NO synthase(iNOS) which is responsible for the production of NO in the vesical tissue of rat Forty Sprauge-Dawley female rats were equally divided into 5 groups. In group 1, normal saline( 0.85 ml/kg) was intravesically instilled one time. In group 2 and group 3, BCG of Pasteur strain(2 mg/kg, normal saline 0.85 ml/kg) was instilled one time and 3 times weekly respectively. In group 4, 10-fold dose of the strain( 20 mg/kg, 0.86 ml/kg) and in group 5, 1/10-fold dose of the strain (0.2 mg/kg, 0.85 ml/kg) were instilled one time respectively. We sacrificed two rats to excise the bladders in each group 1, 3, 7, and 14 days after the instillation( after the last instillation in group 3). iNOS mRNA was not detected in the vesical tissues from the rats of group 1, whereas it was strongly detected in all the vesical tissues from the rats in group 2, 3. or 4. More iNOS mRNA was detected 14 days after the instillation in group 3 than group 2 or 4. In group 5, iNOS mRNA was weakly detected 1, 3, and 7 days and not detected 14 days after the instillation. Our results indicate that intravesical BCG instillation of rat induces the expression of iNOS mRNA in the vesical tissue and suggest that the duration and degree of iNOS mRNA expression is dependent on the dose of BCG and the frequency of the instillations. On the basis of these observations, we conclude that adequate dose and frequency are required for effective treatment of superficial bladder carcinoma in the intravesical BCG therapy.

Urinary Excretion of Nitric Oxide Metabolite after Intravesical BCG Instillation in the Rat / 대한비뇨기과학회지

Inducible nitric oxide(NO) is understood as a tumoricidal molecule. In animal study, urinary NO excretion after intravesical BCG instillation is recently known. In this study, the authors investigated the pattern of urinary nitrite (stable oxidizide form of nitric oxide) excretion according to the dosage of instilled BCG or number of BCG instillation. According to the dosage of instilled BCG(Pasteur strain :8.l5 x 1000000CFU/mg), 32 Sprauge-Dawley female rats were di-vided into 4 Groups(Group I: control, Group II: BCG1/l0X, Group III: BCGlX. Group IV :BCG10X). Also, each Group was subdivided into a one-time instillation subgroup(one time instillation at the first day) and a six-time instillation subgroup( weekly instillation for six weeks). The urinary nitrite in 24 hours' urine was measured quantitatively in each group by Titertek Multiscan MCC/340 ELISA Reader on an every other day basis after BCG instillation until decreased to control level. Intravesical BCG instillation of the rats led to significant urinary nitrite excretion in Group II (BCG 1/10X:one-time ;30+/-12, six-time ;52+/-6), Group III(BCG 1X,57+/-8, 62+/-2uM/L), Group IV (BCG 10X;65+/-2, 80+/-5uM/L) compared to Group I (control;22+/-4, 21+/-3uM/L) respectively( p<0.05). There was no statistical difference in the increment of urinary nitrite excretion between Group II, Group III and Group IV. The urinary nitrite excretion increased significantly in Group II and Group IV of six-time instillation subgroup compared to one-time instillation subgroup(p <0.05). BCG instillation resulted in significant excretion of urinary nitrite at the first day, which reached peak urinary level at the period of the 3rd to 9th day and the 11th day after instillation in one-time and six-time instillation subgroup respectively. The excretion also remained increased for 33 days and 44 days in two subgroups respectively. The preservation of the urine at -40'C for 4-weeks had no influence on the urinary nitrite concentration. The study demonstrates the pattern of urinary excretion according to the dosage of instilled BCG or number of instillations in addition to disclosing good effects of BCG on the excretion of urinary nitrite in rats. The following can be deduced from this study. Intravesical BCG instillation using only one tenth of the usual dose of which the BCG is used for treatment of superficial bladder tumor, and six-times resulted in effective excretion of urinary nitrite in rats.

Secretion and production of reactive nitrogen intermediates in urine and peritoneal macrophages after intravesical BCG instillation in the rat / 대한비뇨기과학회지

Cells or the monocyte-macrophage lineage are known to exhibit tumoricidal activity following stimulation by BCG, interferon -gamma (INF-gamma) or bacterial products such as lipopolysaccharide(LPS). While the mechanisms involved remain obscure, the generation of reactive nitrogen intermediateds (RNI) by activated macrophage is considered a major participant in mediating the tumoricidal effect. In this study, the authors intended to know the effects of BCG infection on the production and secretion of RNI in the experimental animals. Sprauge-Dawley rats were instillated with BCG intravesically. The production of RNI from peritoneal macrophages and urinary secretion of RNI were measured after intravesical BCG instillation of the rats. The urinary concentration(micrometer/L) of nitrite, stable oxidized form of nitric oxide(N0-), 1 week after intravesical BCG instillation was 20+/-0.5 in the group I (control). 54+/-1.0 in group II (BCG 1x). 63+/-0.5 in group III (BCG 10x) and 17+/-0.5 in group IV (BCG 10x + N(G)MMA). The urinary nitrite concentration(micrometer/L) 3 weeks after intravesical BCG instillation was 17+/-2.0 in group I, 124+/-3.0 in group II, 210+2.5 in group III and 31+/-0.5 in group IV. The production of RNI by peritoneal macrophages 3 weeks after intravesical BCG instillation increased in group III (45+/-2.0 micrometer/L) compared to group I (5+/-1.0 micrometer/L). The peritoneal macrophages treated with LPS and INF-gamma increased nitrite production (36+/-0.5 in group I , 52+/-1.5 micrometer/L in group III). The production of RNI by peritoneal macrophages was inhibited by the treatment of the rats with N(G)MMA (19+/-0.5 in group 1, 17+/-1.5 micrometer/L in group III). The results of this study showed that BCG infection of the rat via intravesical instillation makes the peritoneal macrophages produced RNI and increases the secretion of RNI in the urine. This study suggest that the effects of BCG infection for the treatment of bladder cancer might be mediated by the production or RNI in the tumor bearing host.