Yoshiki Kuroda

Induction of lupus autoantibodies by adjuvants

Exposure to the hydrocarbon oil pristane induces lupus specific autoantibodies in non-autoimmune mice. We investigated whether the capacity to induce lupus-like autoimmunity is a unique property of pristane or is shared by other adjuvant oils. Seven groups of 3-month-old female BALB/cJ mice received a single intraperitoneal injection of pristane, squalene (used in the adjuvant MF59), incomplete Freunds adjuvant (IFA), three different medicinal mineral oils, or saline, respectively. Serum autoantibodies and peritoneal cytokine production were measured. In addition to pristane, the mineral oil Bayol F (IFA) and the endogenous hydrocarbon squalene both induced anti-nRNP/Sm and -Su autoantibodies (20% and 25% of mice, respectively). All of these hydrocarbons had prolonged effects on cytokine production by peritoneal APCs. However, high levels of IL-6, IL-12, and TNFalpha production 2-3 months after intraperitoneal injection appeared to be associated with the ability to induce lupus autoantibodies. The ability to induce lupus autoantibodies is shared by several hydrocarbons and is not unique to pristane. It correlates with stimulation of the production of IL-12 and other cytokines, suggesting a relationship with a hydrocarbons adjuvanticity. The potential to induce autoimmunity may complicate the use of oil adjuvants in human and veterinary vaccines.

DNA Repair Gene hOGG1 Codon 326 and XRCC1 Codon 399 Polymorphisms and Bladder Cancer Risk in a Japanese Population

BACKGROUND Bladder cancer is the most common urologic malignancy in the USA. Tobacco smoking generates oxidative DNA damage and induces bladder cancer. Base excision repair (BER) is a very important mechanism for repairing oxidative DNA damage. There are many enzymes involved in BER. Human oxoguanine glycosylase 1 (hOGG1) and X-ray repair cross-complementing 1 (XRCC1) are enzyme genes of BER. Actually, the hOGG1 codon 326 polymorphism was associated with the risk of lung oesophagus and stomach cancer. On the other hand, among several XRCC1 gene polymorphisms, codon 399 polymorphism was reported to reduce the risk of bladder cancer and raise the risk of lung cancer. METHODS We examined the association between the genetic polymorphisms of hOGG1 codon 326 and XRCC1 codon 399 and bladder cancer risk. In this study, we recruited 251 bladder cancer cases and 251 healthy controls to evaluate the effect of hOGG1 codon 326 and XRCC1 codon 399 polymorphisms on bladder cancer. We detected genotypes by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS The frequencies of the hOGG1 codon 326 genotypes Cys/Cys was significantly higher in the cases than in the controls. Adjusted odds ratio (OR) was 1.85 (95% CI: 1.12-3.03; p = 0.02) compared with Ser/Ser, and was 2.05 (95% CI: 1.36-3.08; p = 0.01) compared with Ser/Ser + Ser/Cys. In addition, when evaluated with smoking status, the adjusted OR (Cys/Cys versus Ser/Ser + Ser/Cys) ran up to 2.78 (95% CI: 1.39-5.60; p < 0.01) among non-smokers. For the XRCC1 polymorphism, the Gln/Gln of XRCC1 codon 399 genotype was statistically higher in the controls than in the cases though compared with Alg/Alg + Alg/Gln. The adjusted OR was 0.45 (95% CI: 0.21-0.99; p = 0.05), and was lifted up to 0.37 (95% CI: 0.14-0.98; p = 0.05) among smokers. CONCLUSION It is indicated that the hOGG1 codon 326 and XRCC1 codon 399 polymorphisms are risk factors of bladder cancer.

Prostaglandin E2 receptors EP2 and EP4 are up-regulated in peritoneal macrophages and joints of pristane-treated mice and modulate TNF-α and IL-6 production

Prostaglandin E2 (PGE2) can have pro‐ or anti‐inflammatory effects, depending on engagement of different PGE2 receptor (EP) subtypes. The role of EPs in regulating autoimmune inflammation was studied in the murine arthritis/lupus model induced by pristane. Peritoneal macrophages were isolated (biomagnetic beads) from BALB/c, DBA/1, or C57BL/6 mice treated with pristane (intraperitoneally, 3 months earlier) or thioglycolate (3 days earlier) or with untreated controls. EPs, inducible nitric oxide synthase (iNOS), and cyclooxygenase‐2 (COX‐2) mRNA expression was examined by reverse transcriptase‐polymerase chain reaction (RT‐PCR). Cells were cultured unstimulated or stimulated with lipopolysaccharide (LPS) or LPS + interferon‐γ in combination with EP subtype‐specific agonists. Tumor necrosis factor α (TNF‐α) and interleukin (IL)‐6 production was tested by enzyme‐linked immunosorbent assay (culture supernatant) and flow cytometry. TNF‐α mRNA levels also were examined. High levels of EPs (EP4/2>EP1>EP3), iNOS, and COX‐2 mRNA were expressed in peritoneal macrophages from pristane‐treated but not untreated or thioglycolate‐treated mice (RT‐PCR). TNF‐α production was inhibited 50–70% at 2–24 h by EP4/2 agonists, whereas IL‐6 was enhanced up to ∼220%. TNF‐α inhibition is mediated partly via the protein kinase A pathway and partly via IL‐6. Intracellular TNF‐α staining was inhibited 20% by EP4/2 agonists. TNF‐α mRNA levels were inhibited 50–70% at 2–24 h, indicating that TNF‐α inhibition was partly at the level of transcription. EP1/3 agonists had little effect. Synovial cells from mice with pristane‐induced arthritis (DBA/1) also expressed EP2/4, and the EP2/4 agonist inhibited TNF‐α production. PGE2 can modulate inflammatory reactions via the EP2/4 receptor through its regulation of TNF‐α and IL‐6. Modification of EP signaling may be a new therapeutic strategy in inflammatory/autoimmune diseases.

Protective effect of rutin against spatial memory impairment induced by trimethyltin in rats

Rutin is a flavonoid with various biological activities that are beneficial to human health. Trimethyltin is a toxic organotin compound, and rats injected with trimethyltin serve as a useful in vivo model for studying spatial memory impairment and neurodegeneration in the hippocampus. The protective effect of rutin against the trimethyltin-induced spatial memory impairment and hippocampal neuron damage in rats was examined. Peroral administration of a single dose of trimethyltin (8.5 mg/kg) induced spatial memory loss and the extensive loss of CA3 pyramidal neurons in hippocampi, as indicated by the results of a Morris water maze task and histologic examination, respectively. Prolonged supplementation of rutin significantly reversed the trimethyltin-induced spatial memory impairment and the damage to pyramidal neurons in the hippocampal CA3b region, indicating an antioxidative effect of rutin. These results suggest that rutin in the diet may provide a protective effect against spatial memory impairment accompanied by hippocampal pyramidal neuron loss.

p53 Codon 72 polymorphism and urothelial cancer risk.

The p53 tumor suppressor gene is often mutated in various human cancers. Recently, the p53 codon 72 polymorphism has been extensively studied to determine the risk factors responsible for cancer formation. We investigated the genotype distribution of the p53 codon 72 polymorphism in 112 male urothelial cancer cases and 175 male unrelated non-cancer controls. The allelic frequencies in Japanese non-cancer controls were 0.58 (Arg) and 0.42 (Pro). There was no significant difference in the three genotype frequencies (Arg/Arg, Arg/Pro, Pro/Pro) of the p53 codon 72 between the urothelial cancer cases and the controls. However, stratifying by smoking status, we found that the frequency of the Pro/Pro genotype for smokers was significantly more than that for never-smokers (odds ratio (OR)=2.28, 95% confidence interval (95%CI)=1.12-4.66). Furthermore, we divided smoking status (pack-years) into quartiles (<20, 20-40, 40-60, >60). OR (Pro/Pro vs. Arg/Arg) for the lighter smokers (<20 pack-years) was higher than in other groups (OR=6.83). Our results suggest that the Pro/Pro genotype of the p53 codon 72 polymorphism increases the risk of urothelial cancer in smokers.

Association of genotypes of carcinogen-activating enzymes, phenol sulfotransferase SULT1A1 (ST1A3) and arylamine N-acetyltransferase NAT2, with urothelial cancer in a Japanese population.

Carcinogenic aromatic amines such as 4‐aminobiphenyl, which is contained in tobacco smoke, are one of the causal factors of urothelial epithelial cancers. 4‐Aminobiphenyl has been shown to be bioactivated through N‐hydroxylation by hepatic cytochrome (CYP) 1A2 and subsequently through O‐sulfation and O‐acetylation by phenol sulfating sulfotransferase, ST1A3 (SULT1A1), and arylamine N‐acetyltransferase, NAT2, respectively. In a case‐control study for urothelial epithelial cancers, low activity alleles of NAT2 are overall high‐risk alleles (OR 2.11; 95% CI 1.08–4.26). Wild‐type ST1A3*1 (213Arg) alleles were slightly overrepresented in nonsmoking urothelial cancer patients (82.6% vs. 69.7%) and in smoking cancer patients (76.7% and 74.3%) compared to a variant ST1A3*2 (213His) allele. In combination of ST1A3 and NAT2 genotypes for analyses of urothelial cancer risk, the highest OR of 2.45 (95% CI 1.04–5.98) was obtained with ST1A3*1 and NAT2 slow genotype among the 4 combinations. Recombinant ST1A3*1 enzyme showed a tendency of catalyzing higher in vitro 3′‐phosphoadenosine 5′‐phosphosulfate‐dependent DNA adduct formation than ST1A3*2 (2.84 ± 0.49 and 2.22 ± 0.11 adducts/108 nucleotides). Combined analyses of different alleles of carcinogenic aromatic amine‐activating phase II enzymes were applied to urothelial cancer risk for the first time and showed the highest risk combination of ST1A3 and NAT2 alleles.

E-cadherin gene polymorphism and risk of urothelial cancer.

The E-cadherin is important in cell-cell adhesion and in the development and maintenance of the epithelial phenotype. A -160C-->A polymorphism in the promoter region of E-cadherin has been shown to decrease gene transcription. This allelic variation may be a potential genetic marker that can help identify those individuals at higher risk for invasive/metastatic disease. We studied the effect of E-cadherin gene polymorphism on urothelial cancer susceptibility in a case control study of 314 urothelial cancer patients and 314 age-sex matched controls, to determine whether this polymorphism is a biomarker for the risk and how aggressive the disease is. The frequency with which the subjects carried E-cadherin A/A genotype was significantly higher in the urothelial cancer patients than in the healthy control subjects (OR=2.32, 95% CI 1.03-5.22). Subdividing urothelial cancer according to tumor differentiation and stage, we found no association between E-cadherin polymorphism and poorly-differentiation and invasiveness of urothelial cancer. Furthermore, no significant association between E-cadherin polymorphism and recurrence rate of urothelial cancer patients was found. The present study demonstrates for the first time that E-cadherin A/A genotype may be associated with susceptibility to urothelial cancer, but not with the progression of disease.

Cytochrome P450 (CYP) 1A2, sulfotransferase (SULT) 1A1, and N-acetyltransferase (NAT) 2 polymorphisms and susceptibility to urothelial cancer

Purpose Arylamines are suspected to be the primary causative agent of urothelial cancer in tobacco smoke. In the human liver, arylamines are N-hydroxylated by a cytochrome P450 (CYP)1A2-catalyzed reaction, which produces a substrate for O-esterification that can be catalyzed by N-acetyltransferases (NAT) or sulfotransferases (SULT). Recently, several polymorphisms of CYP1A2, SULT1A1, and NAT2 that affect their activities have been reported.Methods In this study, 306 Japanese patients with urothelial transitional cell carcinoma and 306 healthy controls were compared for frequencies of CYP1A2, SULT1A1, and NAT2 genotypes.Results The frequencies of NAT2 intermediate or slow acetylator genotype were significantly higher in the urothelial cancer patients than in the healthy control subjects [odds ratio (OR)=1.49, 95% confidence interval (95% CI) 1.06–2.09, OR=3.23, 95% CI 1.72–6.08, respectively]. Stratifying by amount of smoking, among subjects who consumed >33.5 pack-years and carried the SULT1A1 *1/*1 or NAT2 slow acetylator genotype, the OR was 1.73 (95% CI 1.01–2.97) whereas it was 7.31 (95% CI 1.90–28.05) in non-smokers who carried the homozygous wild genotype, respectively. The relationships between CYP1A2, SULT1A1, and NAT2 polymorphisms and clinical findings including tumor differentiation, stage, and recurrence rate were analyzed. Only associations between NAT2 genotype and pathological findings were admitted, and the higher OR of NAT2 intermediate and slow acetylator genotype was more likely to present to a low-grade tumor (G1) among heavy-smokers.Conclusions Our results suggest that SULT1A1 *1/*1 and NAT2 slow acetylator genotypes might modulate the effect of carcinogenic arylamines contained in tobacco smoke, and that the modulation of NAT2 intermediate and slow acetylator genotype has a tendency to present a higher risk for highly differentiated tumors among heavy-smokers.

Lack of Evidence for the Association of E-Cadherin Gene Polymorphism with Increased Risk or Progression of Prostate Cancer

Background: E-cadherin is an epithelial cell adhesion molecule, and decreased E-cadherin expression in human prostate cancer is associated with tumor grade and advanced clinical stage. A –160 C→A polymorphism in the promoter region of E-cadherin has been shown to decrease gene transcription. This allelic variation may be a potential genetic marker that can help identify those individuals at higher risk for invasive/metastatic disease. Materials and Methods: We studied the effect of E-cadherin gene polymorphism on prostate cancer susceptibility in a case control study of 219 prostate cancer patients and 219 male controls, to determine whether this polymorphism is a biomarker for the risk and how aggressive the disease is. Results: The genotype frequencies in the prostate cancer group were C/C: 0.607, C/A: 0.352, A/A: 0.041, and in the control group C/C: 0.671, C/A: 0.301, A/A: 0.027. A significant difference between the two groups was not found (p = 0.34), and the adjusted OR for A/A genotype was not statistically significant (OR = 1.66, 95% CI 0.58–4.78). Subdividing prostate cancer according to tumor differentiation and stage, we found no association between E-cadherin polymorphism and poor differentiation and invasiveness of prostate cancer. Conclusions: These data do not support an association between the E-cadherin genotype and the occurrence or progression of prostate cancer in Japanese populations.

Effects of cytochrome P450 (CYP) 2A6 gene deletion and CYP2E1 genotypes on gastric adenocarcinoma

Cytochrome P450 (CYP) 2A6 and CYP2E1 are enzymes with a high ability to activate a nitrosamine, 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK), to its potent and ultimate carcinogens. The polymorphic CYP2A6 and CYP2E1 have been implicated in increased susceptibility to certain malignancies. In our study, 120 Japanese patients with gastric adenocarcinoma and 158 healthy controls were compared for frequencies of CYP2A6 and CYP2E1 genotypes. The frequency with which the subjects carried homozygotes of the CYP2A6 gene deletion allele, which causes lack of the enzyme activity, was significantly higher in the gastric cancer patients than in the healthy control subjects (OR = 3.14, 95% confidence interval (95% CI) = 1.05–9.41). Subdividing gastric adenocarcinoma according to tumor differentiation, patients with the well‐differentiated type were 4.9‐fold more likely to have the CYP2A6 homozygote deletion genotype (OR = 4.91, 95% CI 1.17–20.52). Stratifying by smoking status, we did not find the risk of CYP2A6 gene deletion allele in gastric adenocarcinoma. The CYP2E1 polymorphism detected by RsaI was not significantly different between gastric adenocarcinoma patients (40.8%) and the control population (44.3%). No statistically significant changes were observed when the CYP2E1 genotype was examined relative to tumor differentiation and smoking status. These results suggest that the CTY2A6 deletion is associated with gastric adenocarcinoma among Japanese populations.