Cigarette smoke contains more than 5,000 harmful chemicals, among which nicotine is one of the most dangerous substances and the most abundant in the tobacco leaf. Nicotine is addictive in humans because a portion of the nicotine molecule is similar to acetylcholine, an important brain neurotransmitter. Additionally, it has been found to be present in high concentrations in the bloodstream and urine of smokers (1). Cigarette smoking (CS) is a major risk factor for bladder cancer and according to epidemiological data, smokers, regardless the gender, have two times higher possibility to develop this type of cancer (1). CS is a major risk factor also for bladder dysfunctions such as incontinence and poor bladder and urethral contraction. Furthermore, CS induces the overproduction of reactive oxygen species (ROS) which can damage nucleic acids, proteins and lipids. ROS are considered as a significant class of carcinogens participating in cancer initiation, promotion and progression. Moreover increased ROS induced by bladder outlet obstruction or chronic bladder ischemia have been demonstrated to significantly affect the bladder dysfunction. In the present study, as a major addictive substance of cigarette smoke we selected nicotine and we investigated the effects of nicotine-induced alterations in oxidative stress in the rat bladder. Additionally, the effects of abstinence in the expression of these oxidative stress markers in the bladder were investigated.

Adult male rats were exposed to nicotine dissolved in drinking water for 10 weeks (100μg/mL; Nico group; n=10). Another group was treated with nicotine dissolved in drinking water for seven weeks (100μg/mL) followed by three weeks of abstinence (Abst group; n=10). The control group (n=10) had free access to drinking water during the experimental period which lasted 10 weeks. At the completion of the 10-week experimental period the animals were sacrificed and the bladders were collected. The oxidative stress parameters: 4-hydroxynonenal (4-HNE); malondialdehyde (MDA); 8- hydroxyguanonisine (8-OHdG) were evaluated in the bladder by immunohistochemistry (IHC); 4-HNE and MDA were used as reliable markers for the evaluation of lipid peroxidation, while 8-OHdG  was used as a markers of oxidative DNA damage. Cotinine, a metabolite of nicotine, was measured in the serum and the urine. The expression and localization of nerve growth factor (NGF), which has been identified as a key protein for bladder dysfunction, was investigated by IHC.

Ten weeks of nicotine treatment resulted into significant decrease in the body weight (BW), bladder weight (BL) and the ratio BL/BW in Nico group compared to Control. Abstinence for three weeks could significantly increase the BL in the Abst group compared to Nico group. On the other hand, abstinence significantly decreased the cotinine levels in the serum and urine  in the Abst group compared to the Nico group. IHC revealed strong expression of lipid peroxidation marker 4-HNE localized both in the urothelium and the smooth muscle cells of the bladder in Nico group, while in Control and Abst groups the expression was mild and localized mainly on the urothelium. MDA showed moderate to strong expression in the urothelium of Nico group, while DNA oxidative damage marker 8-OHdG was strongly expressed in both urothelium and muscle cells area in Nico group compared to the other two groups. NGF expression in the urothelium of Nico group was moderate compared to poor expression in the other two groups. Furthermore histological alterations were observed in the bladder tissue of the Nico group such as: damage in the urothelium indicated by the abruption of the lamina propria from the transitional epithelium, degeneration of the submucosa and edema in the substantial epithelium. These histological changes were not present in the bladder tissue samples of Abst group, where the histological pattern was closer to the Control group.

The CS-induced alterations in the bladder function are still not clear. There are some studies demonstrating an association between CS with lower urinary tract symptoms (LUTS) (2,3), but there are still many aspects that need to be clarified. In the present study we investigated the bladder tissue alterations induced by oxidative stress in a nicotine-treated rat model. The nicotine treatment via the upregulation of the oxidative stress markers in the bladder both in the urothelium and the smooth muscle area, further led into an increased expression of the key protein for the bladder dysfunction, NGF. This is a first step to shed some light into the mechanism through which CS induces to some level bladder dysfunction. Future studies which will quantify the expression of NGF and include functional studies are necessary to give clear answers for the exact mechanism through which CS may cause bladder dysfunction.

Nicotine treatment for 10 weeks induced upregulation of lipid peroxidation and DNA oxidative stress, all elements of carcinogenesis initiation or bladder dysfunction. Abstinence from nicotine for three weeks could decrease these parameters. Urologists need to inform their patients about the relationship between smoking and urologic disease, and motivate them to participate in a smoking cessation program as part of their treatment.

David Mobley, MD1 and Neil Baum Smoking: Its Impact on Urologic Health Rev Urol. 2015; 17: 220–225.
Tähtinen RM, Auvinen A, Cartwright R, Johnson TM 2nd, et al. Smoking and bladder symptoms in women. Obstet Gynecol, 2011; 118: 643-648.
Koskimäki J, Hakama M, Huhtala H, & Tammela, TLJ. Association of smoking with lower urinary tract symptoms. J Urol 1996; 1159: 1580-1582.