92 NITROSAMINESANDRELATEDN-NITROSO COMPOUNDS

Challis,

### B. C.;Latif,F., J. Chem.Soc. Perkin Trans 1,1990, 1005-1009. White,E.H.,

### J.Arner. Chem.Soc., 1955,77, 6008-6010. Williams,D.L. H.,Nitrosation, C.U.P.,Cambridge, 1988. Kurosky,A.;Hofmann,T., Canad.

### J. Biochem., 1972,50, 1282-1296. Challis, 

### B. C.;Fernandes, M. H. R.; Glover, 

### B. R.; Latif, F., In The Relevance of N-Nitroso Conzpounds to Human Cancer; Bartsch, H.; O'Neill, 

### I.; Schulte-Hermann, R. Eds.; IARC, Lyon, 1987,IARC Scient. Publ."NO.84, 308-314. Goldstein, 

### E.; Peck, N. F.; Parkes, N. J.; Hines, H. H.; Steffey, E. 0.; Tarkington, B.,Amer. Rev. Respir. Diseases, 1977, 115,403-412. Keise, 

### M., In Methemoglobinemia- A Comprehensive Review, CRC Press, Cleveland, Ohio, 1974,72-73. Doyle, 

### M. R.;Hoekstra,J. W.,

### J.Inorg. Biochem., 1981, 14,351-358. Howard, A. J.; Austin, J., J. Chem. Soc., 1961,3278-3282; ibid.,J. Chem. SOC., 1961,3284-3289. Markgraf,J.H.; Davis,H. A,,J. Chem.Ed., 1990,67, 173-176. Maycock, C.D.; Stoodley,R. J.,

### J. Chem.Soc.Perkin Trans. 1,1979, 1852- 1855. Pollock,J. R. A., Food Chem. Toxicol.,1985,23,701-704. Tricker,A. R.;Preussman, R., Carcinogenesis,1986,7, 1523-1526. Outram, 

### J. R.; Pollock, J. R. A,,In N-Nitroso Compounds: Occurrence, Biological Effects and Relevance to Human Cancer, O;Neill, I. 

### K.; Von Borstel,R. C.; Miller, 

### C. 

### T.;Long, J.; Bartsch,H., Eds.; IARC, Lyon, 1984, IARC. Scient. Pub1.nNo. 57, 71-76. Curtius,T.;Thompson,J., Chem.Ber., 1906,39,4140-4144. Dimroth, O.,Ann., 1910,373,336-346 and referencescited therein. Lawley, P. D, In Chemical Carcinogens 2nd Edn.;Searle,C.E., Ed;ACS monograph 182,ACS,Washington,DC, 1984,428. Meier,I.; Shephard,S.E.; Lutz,W. K., Mutat. Res., 1990,237, 193-197. Long,

### F. A.; Purchase,

### M., J.Amer. Chem.Soc., 1950,72, 3267-3271.

RECEIVED July 28,1993 

## Chapter 

## 8



## Nitrosatable Secondary Amines Exogenous and Endogenous Exposure and Nitrosation In Vivo

A. 

### R. Tricker1, 

### B. Pfundstein, and 

### R. Preussmann

Institute of 

### Toxicology and Chemotherapy, German Cancer Research

Center, 

### Im Neuenheimer Feld 280, D-6900 Heidelberg, Germany

Mean dietary exposure toN-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine 

### (NPYR) and N-nitrosopiperidhe (NPIP) is 0.20-0.3 1 &day; exposure to the secondary amines dimethylamine (DMA), diethylamine (DEA), pyrrolidhe 

### (PYR)and piperidine (PIP) is 7.4 mg/day. These amines are also found in human saliva, gastric juice, blood, urine and faeces. Patient groups with chronic bacterial infections of the urinary tract (paraplegics, Bilharzia and bladder augmentation patients) and healthy controls excreted a mean of 40.5-49.7 mg DMAIday, 19.4-23.8mg PYRIday and 26.1-31.7

### mgPIPiday. Inpatient groups a mean of 0.43-1.88 pg NDMA/day, 0.13-0.39 pg NPWday and 0.21-0.30 pg NPIPIday was excreted in urine but not in a control group. The endogenous formation of amines, and volatile N-nitrosamines under some clinical conditions is considerably higher than the exogenous exposure.

The potential for the endogenous gastric formation of N-nitroso compounds has been evaluated in kinetic models (1) using data obtained from either animal feeding studies (2) or calculations based on the predicted intake of precursor ,amines from the diet 

### (3). These studies suggest that endogenous formation and. exposure to dialkyl and heterocyclic volatile N-nitrosamines is not of sufficient magnitude to present a potential health risk. However, biological monitoring of specific drug nitrosation products formed in man and excreted in urine (4,5) provides strong evidence that endogenous nitrosation can provide a substantial additional exposure to N-nitroso compounds. As inthe case ofN-nitrosodimethylarnine (NDMA) formationfrom endogenous nitrosation ofarninopyrine (4),animal studies show that sufficient endogenous nitrosation occurs for biological monitor-

'~u~~ent address: WissenschaftlicheAbteilung,Verband der Cigarettenindustrie, Konigswintererstrasse550,53227Bonn, Germany

0~-6156/94/0553-0093SO&OO/O @ 1994American Chemical Society 

### This material may be protected by Copyright Law [Title 17 US Code]94 NITROSAMINESAND REL4EDN-mOS0 COMPOUNDS

ing of 7-methylguanine DNA adducts excreted in urine (6) and to induce liver
tumors in rats (7). Theoretical studies suffer hm two major limitations: (i) the lack of available analytical data on the exposure to both nitrosatable amines in the diet &d their

occurrence 

### in biological fluids such as gastric juice, and (ii) the fact that endogenous nitrosation is not limited to chemical reactions involving dietary nitrate, nitrite and amines occurring under acidic conditions in the stomach. Extragastric nitrosationmay alsoresult from reactions involving bacteria (8),yeasts (9), alveolar (10) and peritoneal macrophages (11) in the oral cavity (9), urinary bladder (12,13), sigrnoid colon (14) and possibly the vaginal vault (IS). The following studies evaluate the daily dietary exposure to nitrosatable secondary amines and volatile N-nitrosarnines, and their endogenous occurrence in human saliva, gastricjuice, blood, urine and faeces.



### Methods and Materials

Exogenous exposure to secondary amines and volatile N-nitrosamines. In a dietary survey, 38alcoholic

### drinks and 215foods prepared forhuman comumption using standard culinary practices were analysed for secondary amines (16,17) and volatile N-nitrosamines (18). Food samples were purchased fiom supermarkets 

### in Heidelberg, Germany. The choice of samples was based on the 1988 German Nutritional Report (19) which was used to calculate the mean daily intake of individual foodstuffs. A 10 day duplicate diet survey was also performed, diet samples were collected in plastic containers, homogenized and frozen at -20°C pending analysis.



### Biomonitoring studies. Both smoking and nonsmoking volunteers participating 

### in the studies consumed their normal diets without additional vitamin supplementation. Alcohol consumption was forbidden in investigations in which urine samples were collected. Urine samples were collected &om spine-injured paraplegic patients (13), Bilharzia (12) and bladder augmentations patients (20). A control group was selected from candidates not taking any form ofpharmaceutical drug treatment. Urine (24 hour and individual samples) was collected by direct urination into sterile plastic containers containing 10 g sodium hydroxide. Saliva samples were collected (3-4 hours after break€&) by expectoration into sterile plastic vials containing 10

### M sodium hydroxide (100 pl). Gastric juice samples (5- 10ml) were collected by endoscopy. The pH was immediately measured using a glass electrode prior to stabition by addition of 10 

### M sodium hydroxide (200 pl). Blood (peripheral) samples were collected using heparinized sterile tubes. Plasma was obtained by centrifugation at 3000 g for 15

### mi.at 4°C and stabilized by addition of 10

### M sodium hydroxide (200 pl). Faeces samples (50-100 g) were weighed into plastic bottles containing 0.5 

### M sodium hydroxide (50 rnl) and vigorously shakento produce a homogenous slurry.Sodiumhydroxide stabilization

was used for all samples to prevent artefact N-nitrosamine formation and to prevent bacterial growth andlorbacterial reduction ofnitrate tonitrite. All samples

were stored at -20°C prior to analysis using previously reported methods for the analysis of nitrate, nitrite, secondary amines and volatile N-nitrosamines (21). 

### 8. TRICmR 

## &. NitrosatableSecondaryAmines 95



### Results and Discussion

The exogenous exposure to secondary mines and volatile N-nitrosamines is presented in Table 

### I. Good agreement onthe mean daily intake was obtained from the dietary survey after correction for the daily intake according to the German Nutrition Report and the 10day duplicate diet survey. However, caution must be excercised 

### in the estimation of exposure to food constituents due to uncertainties in the average food consumption within a population. According to the German Nutrition Report, adult women have a total average consumption of 1.8 kg/day while our duplicate diet survey showed an average daily consumption of 2.4 kglday for a 60 kg female adult. The available data fromfood surveysonvolatile N-nitrosamines published over the last decade (reviewed in 18) indicate that: (i) the dietary intake of NDMA is 0.5 &day or less in most countries, and (ii) the current levels are about one-third of those found a decade ago. The latter is true for The Netherlands, Japan and Germany where sufficient data is available to evaluate the dietary situation. This is the first dietary survey which calculates a mean daily intake for secondary amines. Previous estimatesby Shephardet

### al.(3) underestimate the mean exposure to the most commonly encountered amines (1.7 mg DMA/day, 0.3 mg PYRIday and 0.06 mg PIPIday) and overestimate the exposure to other secondary amines (0.6 mg MBzAIday and 0.3 mg MEAlday). The endogenous exposure to nitrate, nitrite and amines expressed in 

### pglrnl (ppm) in saliva, faeces, and blood arepresented inTable 11. Only DMA, PYR and PIP are regularly present in these biological samples. DEA was occasionally found in saliva and faeces but not inblood. Individual samples were occasionally found to contain trace levels of EMA and DBA (data not shown). In the oral cavity, reduction of nitrate to nitrite in the salivary glands (22) produces the highest effective nitrite concentrations in the human body. The presence of oral bacteria, yeasts (9) and increased concentrations of the nitrosation catalyst thiocyanate in the saliva of smokers (23) could theoretically catalyse the nitrosation of secondary amines naturally present in saliva. However, the presence of N-nitrosamines in 'fasting' saliva has not been reported. Ingestion of food increases not only the salivary flow but also increases the levels of nitrite-reactive species in the oral cavity. The concentrations of secondary amines in the diet are
even less than those found in saliva, thus considerably reducing the chance of endogenous nitrosation occurring in the oral cavity during the consumption of a normal diet. Inthe lower gastrointestinal tract,the biological half-life of nitrate (ca. 20 mins) and nitrite (ca. 5 

### mins) is relatively short due to both bacterial metabolism and absorption (24,25). Bacterial decarboxylationof amino acids produces a continuous

source of amines within the gastrointestinal tract (26). These amines are almost completely absorbed (27). Our results show that only low concentrations of secondary amines are excreted infaeces while nitrate and nitrite were not detected. Contrary to an earlierinvestigation by Wang et 

### al. (28) reporting both 

### NDMA and NDEA to be present in normal human faeces, we did not fmd the presence of volatileN-nitrosamines. Theonly reported situation in whichnitrosamine formation

occurs inthe lower intestinal tract is inthe sigmoid colon of ureterosigmoidostomy% MTROSAMINESAND RELATEDN-NlTROSO COMPOUNDS

Table I. Exogenous Exposure to Secondary Arnines andVolatile N-Nitrosamines from the Diet

Mean daily dietary exposure' Food survey 10 Duplicate Men Women diets (women)

Secondary amines (mg/da~)~

DMA PYR 

### PIP DEA 

### DBA MOR 

### EMA DPA 

### MBiA



### NDMA NPIP 

### NPYR

'Calculated horn the 1988 German Nutritional Report 

### (19). 'Secondary amines abbreviated as: Dimethylamine 

### @MA), pyrrolidine (PYR), piperidine (PIP), diethylamine 

### PEA), dibutylamine 

### @BA), morpholine 

### (MOR), methylethy lamine (EMA), dipropylamine (DPA)and methylbenzylamine 

### (MBzA). Volatile N-nitrosamines abbreviated as: N-nitrosodimethylamine 

### (NDMA), Nnitrosopiperidine 

### (NPIP) and N-nitrosopyrrolidine (NPYR). 

# m, not detected. Limit of detection 10 pgkg for amine and 0.05 pgkg for volatile N-nitrosamine analysis. 8. TRICKER

### ET AL. 

### Nitrosatable SecondaryAmines 97

patients (14). In these patients, the bladder is surgically removed and the ureters implanted into the sigrnoid colon producing an enriched mixture containing urinary nitrate and secondary amines together with nitrate-reducing faecal bacteria. In blood, nitrosatable secondary amines and nitratewere detected. Whilewe did
not analyse for nitrosamine due to the small sample volumes collected, a previous study has shown the presence of NDMA in blood of patients with chronic renal failure 

### (29). The presence of 

### NDMA 

### in blood most probably results from exogenous dietary nitrosamine exposure and/or formation and adsorption from the gastrointestinal tractprior to first-pass hepaticmetabolism. However, the possibility that 

### NDMA is formed in blood or tissue as a result ofNO synthase activity cannot be neglected. The presence of nitrate, nitrite and secondary amines in gastric juice as a function of pH is shown in Figure 1.The pH of 50 gastricjuice samples ranged between pH 1.1 and 8.1 of which 56 %were acidic (pH<4.0) and only 

### 2 samples had a pHB7.0 (data not shown). Nitrate was present in all of 50 samples at a mean concentration of 16.0

# + 22.5 pg/ml (range 0.9-72.0 pg/ml), a general decreasing trend in nitrate concentration was observed with increasing gastric pH. Nitrite was detected in 13 of 50 gastric juice samples at a mean concentration of 0.40

# + 1.26 pg/ml (range ND-8.0 pg/ml). Only 

### 2 gastric juice samples with pHC4.0 contained free nitrite (0.1 and 0.6 pg/ml). At pH>4.0, 11 of 32 gastric juice samples contained 0.60 f 1.55 pg/ml (range ND-8.0 

### (~g/ml) nitrite. 

### DMA was found in all 50 gastric juice samples at amean concentration of0.82 f0.82 pglml (range 0.20- 4.20 pglml). Lower concentrations of 0.06 f 0.1 1 pg/ml (range ND-0.45 pglml) DEA were detected in 19 samples, 0.17

# +0.14 pgfrnl(range ND-0.70 pglrnl) 

### PYR in 43 samples and 1.31

### +- 2.44 pg/ml (range ND-15.8 pgtml) PIP in 48 samples. A marked decrease in amine concentration was found with increasing gastric pH. Isolated gastric juice samples contained EMA and DBA at concentrations of less than 0.06 pglml. In the fasting stomach, the only secondary amines of any consequence which

occur 

### in gastric juice are 

### DMA, PYR and PIP. Chemical nitrosation is controlled by two basic physical properties: (i)the dissociation ofnitrite in aqueous media for which the acid-nitrite equilibrium has a pKa of 3.4, and (ii) the basicity of the amine. Protonated amines are unreactive to the nitrite-derived nitrosating agent N20,. As a result, amine nitrosation rates typically show 

### an optimum atpH 3.0-3.5 due to complex interactions of decreasing concentrations of N203and increasing concentrations of free amines with increasing pH (30). For strongly basic second

### ary amines such as PYR (pKa 11.3), PIP (pKa 11.2) and 

### DMA (pKa 10.7), the effective concentration of the unprotonated amine which can participate in chemical nitrosation under acidic conditions is small. At present, the extent of nitrosation in the gastric compartment cannot be assessed. However, endogenous nitrosation of aminopyrine (8) and piperazine (9) 

### in man and the monitoring of their nitrosation products in urine show that a range of amino precursors do undergo nitrosation in vivo resulting in considerable transient levels of 

### Nnitrosamines after exposure to 

### high concentrations of a single nitrosatable amine. The mean 24 hour levels of nitrate, nitrite, secondary amines and their corresponding N-nitroso derivativesin urine of

### three different patient groups prone to chronic bacterial infections of the urinary bact, 

### and in 

### a comparative German98 NITROSAMINESAND RELATEDN-NITROSO COMPOUNDS

Table 11. Nitrate, Nitrite and Secondary Amines in Saliva, Blood and Faeces (@ml)l



### Saliva (n=20) 

### Blood (n=10) Faeces 

### (n=8)

- -- -- 

### NO, 35.5

# + 17.4 3.21 f 

### 2.74 ND2 (5.8-92.0) (1.4-8.1) NO; 8.9 k 6.3 

### ND N-D (1.6-23.0) 

### DMA 0.18

# + 0.05 0.91

# + 0.84 0.41

# + 0.46 (ND-0.30) (0.1 5-2.40) (0.10-1.35) DEA 

### ND 0.03 k 0.04 (0.03) (ND-0.10) PYR 0.21

# + 0.23 0.36

# + 0.58 0.55 k 0.67 (ND-0.65) (ND-2.20) (0.25-1.30) 

### PIP 0.49k 0.37 1.86

# + 1.02 0.39 k 0.24 (ND-1.30) (0.45-2.70) (0.20-0.70)

'Mean fS.D. (rangeofindividual results). Secondary amines abbreviated as shown in Table I. 

### 'ND, not detected. Limit of detection 1.0 pglml for nitrate and nitrite, 0.01 pglml for secondary amine analysis.

Arnine concentration (~g/rnl) 5

Gastricjuice pH 1-1.9 2-2.9 3-3.9 4-4.9 5-5.9 6-6.9

Nitrate (pprn) 28.9 10.7 14.5 19.5 12.3 17.1 Nitrite (pprn) 0 0 0.09 0.15 0.34 0.48 Figure 1.Concentrations ofNitrate, Nitrite and SecondaryArnines in Gastric Juice



### as a Function of pH. 8. TRICKEX ETAL. Nitrosatable SecondaryAmines 99

control group ofhealthy subjects, are given

### in Table 111.Urine contains the highest concentrations ofboth secondary amines and nitratewithin thehuman body. Under normal conditions, in the absence of bacterial infections, nitrosation within the urinary tract does not occur. In the theepatient groups with bacterial colonisation ofthe urinary tract, excretion of

### NDMA, NPIP and 

### NPYR most probably occur as

a result of bacterial mediated nitrosation or as an indirect result of the infection which stimulates NO production. Aerobic bacterial cultures showed a complex

Table 111. Daily Excretion of Nitrate, Nitrite, Secondary Amiies and Volatile 

### NNitrosarnines in Urinei

Patient groups with bacterial infections of the urinary tract

Control Paraplegic Bladder Bilharzia
group patients augmentations patients (n=40) (n=30) (n=1 

### 2) (n=50)



### (0-5) 23.8k15.3 (7.4-45.0) 31.7k17.4 6.6-98.1) 0.43k0.38 (ND-

### 1.40) 

### ND 158.1k166.7 (16.0-855) 3.36k4.02 (ND-45.0) 37.8k17.2 (9.8-80.7) 0.69k1.75 (ND-9.04) 21.9323.2 (1.4-78.8) 24.2K29.7 (0.6-1 17.1) 1.88k4.63 (ND-32.1) 0.10k0.18 (ND-

### 1.90) 0.16k0.25 (ND-0.90) 0.21+0.18 (ND-0.60)

'Mean f S.D. (range of individual results). Secondary amines and volatile 

### Nnitrosamines abbreviated as shown in Table I. 

### 'ND, not detected. Limit of detection 1.0pdml for nitrate and nitrite, 

### 0.01 pg/d for secondary amine, and 0.05 pg/l for volatile N-nitrosamine analysis.100 NITROSAMINESAND RELATEDN-NITROSO COMPOUNDS

bacterial flora including nitrate-reducing bacteria in urine samples from both paraplegic patients and patients with bladder augment-ations. Bacterial cultures were not obtained from Bilharzial patients. However, other studies confii that chronic bacterial infections are usually present in such patients (31). In summary, our results show the concentrations of nitrosatable secondary amine precursors to volatile N-nitrosamines in the diet and in different compartments ofthehuman body in which endogenous nitrosationmay occur. The endogenous formation of these mines is far greater than the exogenous exposure from the diet. The principal site ofamine formation is in the lower gastrointestinal tract from which amines are almost totally absorbed and circulated in blood. From blood, amines may diffuse into acidic gastric juice where on protonation there is little net transport from the gastric lumenback intoblood. As aresult, most amines
enter an enterogastric cycle prior to renal excretion.

Acknowledgements

The authors are grateful to Dr. D. Stickler, School of Pure and Applied Biology, UWIST, Cardiff, Wales, Prof. M. H. Mostaffa, Institute of Graduate Studies and Research, University of Alexandria, Alexandria, Egypt, and 

### Dr. T. Kalble, Urological Department, University of Heidelberg, Germany for collection of biological fluids used in these studies.

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RECEIVEDJuly 19,1993