BIOMONITORING OF N-NITROSO COMPOUNDS, NITRITE AND NITRATE IN THE URINE OF EGYPTIAN BLADDER CANCER PATIENTS WITH OR WITHOUT SCHISTOSOMA HAEMATOBIUM INFECTION Mohamed A. ABDEL MOHSEN 1,2 , Ashraf A.M. HASSAN 2 , Shehata M. EL-SEWEDY 2 , Tousson ABOUL-AZM 3 , Cinzia MAGAGNOTTI 1 , Roberto FANELLI 1 and Luisa AIROLDI 1 * 1 Department of Environmental Health Sciences, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy 2 Medical Research Institute, University of Alexandria, El Hadara, Alexandria, Egypt 3 Department of Urology, Faculty of Medicine, University of Alexandria, Medan El-Khartoum, Alexandria, Egypt The excretion of nitrate, nitrite, apparent total N-nitroso compounds and volatile nitrosamines was measured in 24 hr urine from 61 Egyptians, divided into 4 groups: controls, Schistosoma haematobium-infected patients and bladder can- cer patients with and without a history of schistosomal infection. U rinary nitrate in S. haematobium-infected patients wassignificantly higher than in the other 3 groups. N itrite was below the detection limit of the method ( I0.015 g/mg creatinine) in all but one of the control samples. S. haemato- bium infection significantly increased urinary nitrite to 0.9  1.16 g/mg creatinine (mean  SD , p  0.001). In both blad- der cancer groups, nitrite was about 20 times that in S. haematobium-infected patientswithout bladder cancer. Excre- tion of apparent total N -nitroso compounds paralleled that of nitrite. Overall, a good correlation was observed between these 2 variables (r  0.71, p  0.0001). N -nitrosodimethyl- amine was present in all the samples analyzed. S. haemato- bium infection significantly increased urinary N -nitrosodimeth- ylamine level compared with that of controls(4.02  1.61 and 2.04  2.97 ng/mg creatinine, respectively, p  0.01). Among cancer patients, N -nitrosodimethylamine was higher than in controls only in those with schistosomal infection. The pres- ence of N -nitroso compounds and N -nitrosodimethylamine in the urine of S. haematobium-infected patients both before and after the development of cancer, and the observation that these compounds also occur in bladder cancer patients with no history of schistosomal infection, suggest that these compounds might have a role not only in the initiation of the carcinogenic process, but also in its progression. Int. J. Cancer 82:789–794, 1999.  1999 Wiley-Liss, Inc. Bladder cancer accounts for about 6% of all cancers in men and 2% in women (Landis et al., 1998). Tobacco smoking, occupational exposure to different chemicals such as aromatic amines, and Schistosoma haematobium infection are among the major risk factors (Matanoski and Elliot, 1981). Less established risk factors include the use of cyclophosphamide, phenacetin-based analgesics and artificial sweeteners (Matanoski and Elliot, 1981). The highest bladder cancer rates worldwide have been observed in Egypt. In Alexandria the incidence of this tumor ranks first among all cancers, accounting for 17.5% and 3.6% of all cancer in men and women, respectively (Bedwani et al., 1993). A substantial proportion of bladder cancers are of squamous-cell type (Bedwani et al., 1993; Kantor et al., 1984). An association between bladder cancer incidence and S. haematobium infection has been observed (IARC, 1994; Bedwani et al., 1998; Badawi et al., 1995). The proposed explanations of the mechanism underlying the develop- ment of bladder cancer associated with schistosomiasis include inflammatory reactions induced by the parasite eggs in the bladder mucosa and submucosa, production of carcinogenic tryptophan metabolites or elevated levels of urothelial -glucuronidase (Badawi et al., 1995). Particular attention has been paid to the role of N-nitroso compounds (NOC) that may be formed in the bladder because of bacterial infection, often superimposed on the parasitic disease. According to this hypothesis, bacteria reduce dietary nitrate to nitrite, which reacts with nitrosatable compounds in the urine to give NOC (Hicks et al., 1977). Nitrosation can also be catalyzed by bacteria (Ohshima et al., 1987). NOC carcinogenicity is well established in animals (Druckrey et al., 1967). In humans, besides being proposed as etiological agents of bladder cancer associated with schistosomiasis, NOC are thought to play a role in the induction of cancers of the stomach, esophagus and nasopharynx (Mirvish, 1995). The sources of exposure, endogenous formation and occurrence in body fluids of NOC have been reviewed by Tricker (1997). The carcinogenic activity of NOC arises from their ability to alkylate DNA and form a variety of adducts, including O 6 -alkylguanines, whose presence and persistence have been correlated with their mutagenicity and carcinogenicity (Saffhill et al., 1985). High levels of NOC and their precursors, nitrate and nitrite, have been detected in the urine of S. haematobium- and S. mansoni- infected patients, including those with bladder cancer, thus support- ing the NOC etiological hypothesis for schistosomiasis-associated bladder cancer (Hicks et al., 1977, 1982; Tricker et al., 1989; Mostafa et al., 1994). To date, no study has investigated whether bladder cancer per se has any effect on the urinary excretion of NOC and their precursors. The main aim of the present study was to investigate further the hypothesis that NOC might be the etiological agents for bladder cancer associated with S. haematobium infection, by comparing the urinary excretion of nitrate, nitrite apparent total NOC (ATNOC) and volatile nitrosamines (NA) in controls, S. haematobium- infected patients and S. haematobium-associated bladder cancer patients. We also investigated these variables in bladder cancer patients with no history of S. haematobium infection. These latter had not been taken into consideration in previous studies. MATERIAL AND METHODS Chemicals N-nitrosoproline was purchased from the National Cancer Institute Chemical Carcinogen Reference Standard Repository. N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosobutylpropylamine Abbreviations: ATNOC, apparent total N-nitroso compounds; NA, N-nitrosamines; NDBA, N-nitrosodibutylamine; NDEA, N-nitrosodiethyl- amine; NDMA, N-nitrosodimethylamine; NDPA, N-nitrosodipropylamine; NMor, N-nitrosomorpholine; NOC, N-nitroso compounds; NPip, N- nitrosopiperidine; NPyr, N-nitrosopyrrolidine. Grant sponsor: C.M.T. Italian Ministry for Foreign Affairs, D.G.C.S. Egyptian-Italian Cooperation Project; Grant sponsor: Italian National Research Council; Grant number: 96.00752.PF39. *Correspondence to: Department of Environmental Health Sciences, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy. Fax: +39 02 3546277. E-mail: airoldi@irfmn.mnegri.it Received 9 November 1998; Revised 4 May 1999 Int. J. Cancer: 82, 789–794 (1999)  1999 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer