鄰苯二甲酸酯類化合物在泌尿上皮癌患者曝露增加造成Nrf-2訊息傳遞與尿中擬桿菌目細菌OTU增加

蔡育賢¹、董修廷¹、吳冠諭¹、蔡欣孜¹、蔡宗欣²、

¹國立成功大學醫學院附設醫院 泌尿部；²安南醫院 泌尿科

Phthalate exposure is increased in urothelial carcinoma patients that enhances Nrf-2 signaling transduction and operational taxonomic units of urine Bacteroidales

Yuh-Shyan Tsai¹, Hsiu-Ting Tung¹, Kuan-Yu Wu¹, Hsin-Tzu Tsai¹, Tzong-Shin Tzai²,

Department of Urology¹, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Department of Urology², Annan Hospital, Tainan, Taiwan

Purpose: Urothelial carcinoma (UC) of urinary tract (renal pelvis, ureter, and bladder) is one of most common cancer in Taiwan. There were several risk factors associated with urothelial carcinogenesis including chronic arsenic exposure, aristolochic acid nephropathy, chronic kidney disease, concomitant urolithiasis, and so on. The role of phthalate exposure remains unclear in urothelial carcinoma.

Materials and Methods: A total of 20 bladder UC, 10 chronic kidney disease, and 10 non-cancerous patients were enrolled for urine 8-OHDG (oxidative stress marker), phthalate metabolite measurements using ELISA and HPLC assays and urine microbiota Next generation sequencing (NGS). A total of 10 renal pelvis UC with paired non-cancerous pelvis wall, and renal cortex were investigated for Nrf-2 mRNA RTPCR and whole-exon sequencing. Four UC tissue microarrays (n=60) were used for Nrf-2 immunohistochemical staining. The immortalized SUHVC-1 and 6 UC cells were used for in vitro analysis of Nrf2 and its downstream signaling with and without phthalate exposure using MTT, RT-PCR, and western blotting assays.

Results: Both urine 8-OHdG levels and phthalate metabolites (MECPP, and MCMHP) are significantly higher in bladder urothelial carcinoma patients (p values, 0.01 and 0.02, respectively). The urine 8-OHdG level is associated with the phthalate exposure in Taiwan. Nrf2 mRNA expression is increased in the urothelial cancer cell lines as compared with immortalized SVHUC cells, as well as Nrf2-targeted proteins. In contrast, GULP1 mRNA expression is decreased in the urothelial cancer cell lines. Phthalate exposure enhances Nrf2 mRNA, protein expression, not associated with GULP1 expression in SV-HUC-1 and TCCSUP cells. Renal pelvis UC exhibits higher Nrf2 mRNA expression and immunostaining than adjacent tissues (cortex and pelvis wall). NGS results of renal pelvis UC and paired adjacent renal pelvis wall, cortex showed that discordance between Nrf2 gene alteration and Nrf2 mRNA expression. Urine microbiota in UC patients before and after treatment, as well as non-cancerous patients. The genus Burkholderia in urine was more abundant in the bladder cancer versus the non-cancerous patients, more in patients with recurrence versus without recurrence. The OUT (operational taxonomic units) of urine Bacteroidales are positively significantly with five urine phthalate metabolites (MEHP, MEOHP, MEHHP, MECPP and MCMHP) (all p values < 0.05).

Conclusions: Phthalate exposure is increased in urothelial carcinoma patients that enhances Nrf-2 signaling transduction and OTU of urine Bacteroidales. The association between phthalate exposure and urine microbiota in urothelial carcinogenesis is largely worth to further explore.