11 The Role of Bacteria in Nitrosamine Formation
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D. RALT and S. R. TANNENBAUM Department of Nutrition & Food Science, Massachusetts Institute of Technology, Cambridge, MA 02139
Bacteria have been implicated in the formation of N-nitroso compounds under a wide variety of conditions representing both in vitro and in vivo situations. Mechanisms of participation and/or catalysis include a) decrease of the pH of the system, b) reduction of nitrate to nitrite, c) adsorption of amine onto the cell surface or cytoplasmic membrane, d) actual enzymatic formation. The literature of the field will be reviewed and experimental evidence which tests the above mechanisms will be presented. Recently nitrosamines have attracted attention because of their marked carcinogenic activity in a wide variety of animal species (1, 2). Nitrosamines are likely to be carcinogens in man as well: human exposure to these compounds i s by ingestion, inhalation, dermal contact and in vivo formation from nitrite and amines. Nitrite and amines react most rapidly at an acidic pH. A variety of factors, however, make nitrosation a potentially important reaction above pH 7: these include the presence of microorganisms, and the possibilities of catalysis by thiocyanate, metals and phenols, and of transnitrosation by other nitroso compounds. J. Sander (3) and M. J. Hill and coworkers (4) were among the first to recognize the role that bacteria might play in endogenous nitrosamine formation. Since then several other workers have suggested that microorganisms play a role in N-nitrosation. Hicks et al. (5) and Radomski and coworkers (6) have shown an association of nitrosamines in human urine with chronic urinary tract infections and possibly with bladder cancer. We have shown (7) that oral microorganisms reduce nitrate to nitrite, and that salivary nitrite reacts with secondary amines added to saliva to form the corresponding nitrosamines: the reaction rate is increased in the presence of bacteria. Crissey et al. (8) have 0097-6156/81/0174-0157$05.00/0 © 1981 American Chemical Society Scanlan and Tannenbaum; N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
158
N-NITROSO COMPOUNDS
shown that contact with the fecal stream i s necessary i n rats f o r the experimental induction of colon tumors following ureterosigmoidostomy. I t i s conceivable that nitrate introduced into the fecal stream from the urine i s reduced by bacteria to n i t r i t e , which can react to form N-nitroso compounds i n the colon. Only a few studies have been undertaken on the role of bac t e r i a i n nitrosation at neutral pH i n the i n t e s t i n a l tract i t s e l f . Hashimoto et a l . (9) demonstrated that by changing the equilibrium between different species of microorganisms i n the gut, (by administration of a n t i b i o t i c s and inoculation with nitrate-reduc ing strains) dimethylnitrosamine (NDMA) accumulated i n the stomach and cecum of rats fed dimethylamine (DMA) and n i t r a t e . The authors describe only one experimental group, however, and the mechanism of NDMA formation i s not clear. Other investigators have carried out i n v i t r o experiments using i n t e s t i n a l contents or isolated bacteria i n order to assess the potential role of microorganisms i n the formation of N-nitroso compounds (3, 4^ 10-13). The format of these experiments i s essentially as follows. Bacteria are added to complex medium supplemented with DMA, glucose and n i t r a t e (or n i t r i t e ) ; the u t i l i z a t i o n of DMA and the formation of NDMA are then followed and compared to the l e v e l of NDMA i n the uninoculated control medium. Three problems arise under those conditions: i ) The bacterial reduction of nitrate to n i t r i t e i s very rapid; therefore an appropriate control medium should contain n i t r i t e , not n i t r a t e ; i i ) Bacterial growth ( u t i l i z ing glucose) causes a decrease i n pH even i n buffered media, so control media should be adjusted to the f i n a l pH of the growing culture; and i i i ) In some experiments, the method of detection of NDMA has not been specific (11, 13); therefore differences between culture and medium could have been due to other metabo l i t e s accumulating i n the growing culture. We f e l t that these complications made i t impossible to isolate the role of micro organisms i n N-nitrosation. We therefore i n i t i a t e d carefully controlled experiments designed to determine whether microorgan isms can s p e c i f i c a l l y catalyze formation of NDMA from n i t r i t e and DMA. In order to define the conditions of the growing cultures, buffered medium (VL) inoculated with IS. c o l i ATCC 11775 and sup plemented with n i t r a t e , glucose and DMA was incubated at 37°C, and pH, n i t r i t e concentration, n i t r a t e concentration, c e l l growth and nitrosamine formation were followed (Fig. 1). Within 2 hrs, >90% of the n i t r a t e i s converted to n i t r i t e (some of the n i t r i t e i s further reduced) and over 8 hrs the pH drops from 7.3 to 6.0. This would indicate that i n experiments carried out f o r 20 hrs or more the control medium should be adjusted to pH 6.0 to 6.5 and n i t r i t e should be added rather than n i t r a t e . Such a control medium (VL) was supplemented with n i t r i t e and DMA; and NDMA forma tion was followed (Fig. 2). I t can be seen that even without the addition of c e l l s the rate of nitrosation i s 4 fold greater than
Scanlan and Tannenbaum; N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
RALT AND TANNENBAUM
Bacteria
in Nitrosamine
Formation
159
Figure 1. pH, nitrite, nitrate, and NDMA concentration as a function of cell growth in VL medium. E . coli ATCC 11775 was inoculated into sealed tubes containing VL medium (described in the legend to Table I) supplemented with 0.2% glucose, 0.05% dimethylamine, and 0.08% sodium nitrate. The tubes were incubated at 37°C and cell growth was followed with spectronic 20 (Bausch & Lomb) at 660 nm. Nitrite and nitrate were measured as described in (14) and NDMA detection as described in the legend to Table I.
Scanlan and Tannenbaum; N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
160
JV-NITROSO COMPOUNDS
1 I
1 I
1 1 Ι—ΓΓΊ—
ι"1
PH « 6 4
30
Culture / Medium /
-
2 Ο ζ
/
10
Thtorttieol or Minimol Medium
~
A \s\
1
4
1 11
8
1
1 1
12
16
1
I
20
1 1
24
Hours
Figure 2. Accumulation of NDMA in VL medium at pH 6.4. The pH of VL medium was adjusted to 6.4 with HCl and NDMA formation was followed at 37°C. Nitrite concentration in the medium was 0.2% and dimethylamine 0.05%. Theo retical curve was calculated according to Fan and Tannenbaum (15).
Scanlan and Tannenbaum; N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
11.
RALT AND TANNENBAUM
Bacteria
in Nitrosamine
161
Formation
the rate predicted for chemical nitrosation under these condi tions. This suggests that the complex medium i t s e l f contains catalysts for N-nitrosation. None of the three strains of IS. c o l i or two strains of proteus that we examined, enhanced the rate of NDMA formation above t h i s rate i n the medium alone. When minimal salt medium was used instead of the complex VL, no en hancement of reaction rate was observed, and the NDMA formed was as expected from chemical nitrosation. I t seemed possible that a c a t a l y t i c effect of the bacteria upon nitrosation might be masked by the high l e v e l - 0.2% - of n i t r i t e used i n these experiments, so we repeated the work with lower concentrations of n i t r i t e (Table I ) . The c a t a l y s i s by the medium alone i s considerably greater at these l i m i t i n g con centrations of n i t r i t e , and again the bacteria do not further enhance the rate of nitrosation. Table I Formation of Dime thy lnitrosamine (NDMA) i n VL medium
uM
NDMA theoretical uM
NDMA actual yM
medium
5.79 2.89
0.16 0.04
4.46 0.81
27.9 20.3
medium + cells
5.79 2.89
0.16 0.04
3.11 0.54
19.4 13.5
NaN0
2
actual/theoretical
VL medium, pH 6.4, with or without c e l l s was incubated i n sealed tubes for 10 hrs at 37 °C. NDMA was determined by gas chromato graphy with the Thermal Energy Analyzer as a detector (Thermo Electron Corp., Waltham, MA). The identity of NDMA was confirmed by GC mass spectrometry. VL medium includes: Trypticase (BBL) 1%, Yeast Extract (Difco) 0.5%, Meat Extract (Difco 0.2%, Hemin 0.0007%, Na C0 0.4%, NaCl 0.05%, NH^Cl 0.1%, MgS0WH 0 0.02% and phosphate buffer 1%. The medium was supplemented with 0.05% dimethylamine (DMA) and 0.2% glucose. 2
3
2
These results led us to reexamine the published work on nitrosation i n the presence of bacteria (Table I I ) . In two of the studies (4, JL0) the amount of NDMA formed i s actually less than the amount that can be predicted from theoretical considera tion of the uncatalyzed chemical reaction alone (15). In another study (12), the y i e l d of NDMA i s again s l i g h t l y lower than the predicted chemical y i e l d at the f i n a l pH (6.0) of the growing culture. The work by Kunisaki and Hayashi (13), on the other hand, does indicate that resting c e l l s of E. c o l i Β catalyze
Scanlan and Tannenbaum; N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
Scanlan and Tannenbaum; N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
8.0 6.4
Kunisaki & E. c o l i B Hayashi, 1979
Ralt and Tannenbaum, t h i s report
drops to ~6.0, as i n Figure 1.
Initial^ pH - 7.5
Hashimoto E. c o l i et a l . , 1975 (feces)
6.5
Initial pH = 7
Intestinal E. c o l i
pH
Klubes Intestinal et a l . , 1972 f l o r a
Hawksworth & H i l l , 1971
micro organism
0.04%
0.7%
0.2%
1.4%
0.2%
nitrite/ nitrate conc.
0.05%
0.2%
0.05%
0.3%
0.1%
amine conc.
10
1
72
20
18
Incubation (hrs)
.2
0.7
68
108
1
5
95
41
95
