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Y Fflchigan Matte values-gin! fig This is to certify that the dissertation entitled Catalysis of N-Nitrosamine Formation by Bacteria presented by Yusef Essanusi Ei-Mabsout has been accepted towards fulfillment of the requirements for Ph .0. degree in Food Science fllt/én‘? r profe Date 8/7/8/ I / MS U is an Affirmative Action/Equal Opportunity Iruu'mn’on 0-1277! OVERDUE FINES: 25¢ per day per item RETURNIMS LIBRARY MATERIALS: Place in book return to move charge from circulation records L‘- 113“ ilk-mi: * V‘ ' ~30" all g “a U” © 1981 YUSEF ESSANUSI EL-MASBOUT All Rights Reserved CATALYSIS OF N-NITROSAMINE FORMATION BY BACTERIA By Yusef Essanusi El-Mabsout A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1981 fa I 2 [3“ 7c; (,3: 1"“ ABSTRACT CATALYSIS 0F N-NITROSAMINE FORMATION BY BACTERIA By Yusef Essanusi El-Mabsout This study was initiated to investigate the role of bacteria in N-nitrosamine formation from immediate precursors (dimethylamine and nitrite), and to investigate the possi- bility of N-nitrosamine formation from several other com- pounds (secondary precursors) via the action of selected bacteria. Compounds investigated were trimethylamine, tri- methylamine oxide, choline, phosphorylcholine, lecithin, sarcosine, N-nitrososarcosine, proline and N-nitrosoproline. Each of the following bacterial species were tested for their ability to produce N-nitrosamines from the fore- mentioned precursors: g. 5911 E-2, g. £911 K-lZ, L. bulgari- £33, 5. thermosphactum, 3. putida, 3. putida biotype A, 3. fluorescens, two 3. schuyjkilliensis species and S. aureus. Growing cultures as well as resting cell suspensions of these bacteria were capable of N-nitrosating dimethylamine when incubated in tryptic soy broth (TSB) or 0.2 M phosphate buffer (pH 8.0), respectively, in the presence of 0.25% dimethylamine and 0.05% sodium nitrite. Bacterial catalysis of the N-nitrosation of dimethylamine was shown to increase with increasing cell concentration. Similarly, N-nitrosamine formation increased with increasing concentrations of nitrite Yusef Essanusi El-Mabsout in the incubation medium. E. 5911 E-Z, g. gglj_K—12 and S. aureus were able to form appreciable amounts of N-nitrosodimethylamine (NDMA) when sodium nitrate (0.25%) was used in the incubation medium instead of nitrite. Lower quantities of NDMA were produced from the secondary precursors by various bacterial species used in this study. None of the bacteria were capable of producing N-nitro- sopyrrolidine (NPYR) from proline and nitrite. However, fl. thermosphactum produced traces of NPYR as a result of decarboxylation of N-nitrosoproline Both NDMA and NPYR were detected in two fried Gid-deed samples (salted sundried lamb). The first was treated with refined salt and nitrite, and the second was treated with rock salt. The production of N-nitrosamines in the latter sample indicated that bacteria indigenous to lamb are capable of reducing nitrate impurities in the rock salt to nitrite. ACKNOWLEDGMENTS The author wishes to express his sincere gratitude to his major professor, Dr. J.I. Gray, for his advice and guidance during the course of this study and for his aid in the preparation of this manuscript. Appreciation and thanks are extended to the members of the guidance committee, Drs. A. Pearson and J. Price of the Department of Food Science and Human Nutrition, Dr. E. Beneke of the Department of Botany and Plant Pathology, and Dr. N. Bergen of the Department of Animal Science, for their advice and effort in reading this manuscript. The author is also indebted to Dr. J.R. Brunner for the use of his freeze dryer and to Dr. N. Bergen for his assis- tance and use of his amino acid analyzer. Genuine appreciation is also extended to Mr. A. Mandegere and Ms. S. Cuppett for their technical assistance. Heartfelt appreciation is also offered to Al-Fateh University for financial assistance, which made this study possible. Last, but not least, the author wishes to express his sincere gratitude to his parents, his wife and his children for their patience and support, and to his friends Al-Mohizea and Ereifej for their helpful discussions and enjoyable companionship. ii TABLE OF CONTENTS LIST OF TABLES. LIST OF FIGURES INTRODUCTION. LITERATURE REVIEW Precursors of N-Nitrosamines and the Nitrosation Reaction. . Catalysis and Inhibition of N- Nitrosation Reactions Catalysis of N- Nitrosamine Formation by Micro- organisms Role of Microorganisms in N- Nitrosamine Forma- tion. . . N- Nitrosamine Degradation by Microorganisms Occurrence of N- Nitrosamines in Foods N- Nitrosamines in Cured Meats N- Nitrosamines in Fish and Fish Products. N- Nitrosamines in Dairy Products. . N- Nitrosamines in Other Food Products Precursors and N- Nitrosamine Formation in Foods Amines in Foods . . Fish and fish products. Meat and meat products. Dairy products. . . Amines in other foods and alcoholic and non- -alcoholic beverages Biologically active amines in foods Nitrates and Nitrites in Foods. In Vivo N-Nitrosamine Formation Carcinogenicity of N-Nitrosamines Gid-deed Preparation. EXPERIMENTAL. Materials Methods Bacteriology. . N- Nitrosation of Dimethylamine with Nitrite Page vi viii Page N- Nitrosation of Dimethylamine with Nitrate. . . . . 43 Decarboxylation of N- Nitrosoproline and. N- Nitrososarcosine . . . . . 43 NDMA Formation from Other Precursors . . . 44 Effect of Incubation Time and Temperature on NDMA Formation by Growing Bacterial Cultures . . . . . . . . . . . . . 44 Resting Cell Studies . . . . . . . 45 Preparation of resting cells . . . . 45 Effect of cell concentration in NDMA formation. . . . . . . . . 45 Protein determination. . . . . . 46 Effect of nitrite concentration on NDMA . . . . 46 Extraction of N- Nitrosamines from Bacterial Cultures . . . . . . . . . . . . . . . . . 46 GC- TEA Analysis. . . . . . . . . . . . . . 47 GC Operating Conditions. . . . . . . . . . . . 48 TEA Conditions . . . . 48 Confirmation of the Identity of N- .Nitrosa- mines. . . . . . . . . . . . . . 49 GC- MS Analysis . . . . . . . . . . . . . . 49 GC Conditions. . . . . . . . 49 Mass Spectrometer Conditions . . . . . 50 UV Irradiation . . . . . 50 N- Nitrosation of Sarcosine by Bacteria and Detection of N- Nitrososarcosine by High Performance Liquid Chromatography (HPLC) . . 50 Gid- deed Processing. . . 52 Free Amino Acid Analysis of the Meat Samples . 53 Vacuum Distillation and Extraction of N- Nitro- samines in the Gid-Deed Samples. . . . . . . 54 Nitrite Analysis . . . . . . . . . . . . . . . 54 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 56 Separation and Identification of N-Nitrosamines by GC- TEA . . , 56 Confirmation of the Identity of N- Nitrosamines by GC- MS Analysis and UV Irradiation. . . . 59 NDMA Formation by Growing Bacterial Cultures from Dimethylamine and Nitrite. . . . . . . 59 NDMA Formation by Resting Cells at pH 8.0.. . . 54 Effect of Nitrite Concentration on NDMA Formation. 55 Effect of Cell Concentration on NDMA Formation . . 58 NDMA Formation by Growing Bacterial Cultures from Dimethylamine and Nitrate. . . . . . . . . . . . 59 iv Page Effect of Incubation Time and Temperature on NDMA Formation by P. schuylkilliensis Growing Cul- tures. . . . 7‘ NDMA Formation by Growing Bacterial Cultures frOm Trimethylamine and Trimethylamine- N- Oxide. . . . 73 Mechanism of NDMA Formation for Tertiary Amines. . 75 NDMA Formation by Growing Bacterial Cultures from Choline and Choline-Containing Compounds . . . . 77 Mechanism of NDMA Formation From Choline and Choline- Containing Compounds . . . . 80 NPYR Formation from L- Proline and N- Nitrosopro- line . . . . 80 NDMA Formation by Growing Bacterial Cultures frOm Sarcosine and Nitrite. . . 84 Detection ofN— NitrososarcosiOneTby High Performance Liquid Chromatography. . . . . 85 Formation of NDMA from N- NitrOsOsarcosine. by Growing Bacterial Cultures . . . . . . . . 88 Identification Of Unknown N- Nitrosamine. . . . . . 39 Precursor Of NDBA. . . . . . . . . 92 Preparation and Analysis Of .Gid- deed . . . 92 Residual Nitrite and N- Nitrosamines in Gid- deed. . 92 Precursors Of NPYR and NDMA in the Fried GiM deed. 97 Free Proline and Free Sarcosine Content of Fresh LambandGid-deed................99 SUMMARY AND CONCLUSIONS... . .. .. .. .... ..102 PROPOSALS FOR FUTURE RESEARCH. . . . . . . . . . . . . 106 BTBLIOGRAPHY.....................108 Table 10 ll LIST OF TABLES Optimum pH and rate constants for the N-nitro- sation of some secondary amines and amino acids. N-Nitrosamines detected in various cured meat products N-Nitrosamines reported in fish and other seafoods N- Nitrosamines (pg/kg) detected in selected dairy products . . . . . . . . Some of the amine compounds reported to be present in fish and fish products. NDMA formation by growing bacterial cultures in 200 ml of tryptic soy broth containing dimethy- lamine (0. 5 g) and nitrite (0. l g) . . . NDMA formation by restin bacterial cells in l00 ml phosphate buffer (0. 2 M, pH 8.0) con- taining 0. 25 g dimethylamine and 0.05 g sodium nitrite. . . . . Effect of nitrite concentration on NDMA forma- tion by S. aureus using a fixed dimethylamine concentration at 37° C and pH 8.0 for 5 hours Effect of cell concentration of E. coli K-lZ on NDMA formation at constant pH (8.0) using fixed concentrations of nitrite and dimethylamine at 37°C for 5 hours . . . . . . . . . NDMA formation by growing bacterial cultures from sodium nitrate (0.5 g) and dimethylamine (0.25 g) in 200 ml TSB . . . . Effect of incubation time and temperature on NDMA formation by P. schuylkilliensis growing in 200 ml of TSB medium containing 0. l g nitrite and 0.5 g dimethylamine . . . . vi Page 21 22 24 26 62 65 67 68 69 72 Table 12 l3 14 15 l6 17 18 19 NDMA formation by growing bacterial cultures from triamines (0. 5 g) and nitrite (0.1 g) in 200 ml tryptic soy broth. . . . NDMA formation by growing bacterial cultures in 200 ml of TSB containing 0.05% (w/v) nitrite and 0.25% (w/v) of each of the choline- -containing compounds. . . . . . . . . . The effect of growing bacterial cultures in NPYR formation from N-Nitrosoproline (0.2%, w/v) in 200 ml of TSB medium . . . . . . . . . . . NDMA formation by growing bacterial cultures from sarcosine (0. 5 g) and sodium nitrite (0. l g) in 200 ml of TSB medium . . . . . . . Determination of N-nitrososarcosine in bacterial cultures by HPLC N-Nitrosamine levels (pg/kg) in raw (salted-sun- dried) and oil fried gid-deed. . . . . Residual nitrite (mg/kg) in raw (salted-sundried) gid-deed . . . . . . . . . . . . Free proline and free sarcosine in fresh lamb and raw (salted-sundried) gid-deed vii Page . 75 . 78 . 84 . 85 . 87 . 93 . 95 . 99 LIST OF FIGURES Figure Page 1 GC-TEA chromatogram of standard N-nitrosodi- methylamine and N-nitrosopyrrolidine. . . . . . . 57 GC-TEA chromatogram of a typical methylene chloride extract of a bacterial culture con- taining precursors of N-nitrosodimethylamine. . . 58 Mass spectra of: (A) N-nitrosodimethylamine standard, and (B) N-nitrosodimethylamine isolated from bacterial cultures containing dimethylamine and nitrite/or nitrate. . . . . . . . . . . . . . 5] Biological degradation of lecithin (adopted from Anonymous, Metabolic Pathways, 1968). . . . . . . 8] Suggested pathway for N-nitrosamine formation from nitrite and choline degradation interme- diates. . . . . . . . . . . . . . . . . . . . . . 82 GC-TEA chromatograms of a standard N-nitrosodi- butylamine and N-nitrosodimethylamine (A), Pseudomonas culture methylene chloride extract (B), and Pseudomonas culture extract plus stan- dard N-nitrosodibutylamine (C). . . . . . . . . . 9] viii INTRODUCTION Historically, the study of the chemistry of aliphatic N-nitrosamines began in 1863 when Geuther obtained N-nitroso- diethylamine (NDEA) by the reaction of diethylamine hydro- chloride with sodium nitrite (Crosby and Sawyer, 1976). Twelve years later, Fischer (1875) reported that N-nitroso- dimethylamine (NDMA) was formed in high yield when dimethyl- amine hydrochloridevms acidified with sulfuric acid and treated with sodium nitrite in an aqueous medium. The present interest in N-nitrosamines started in 1956 when Magee and Barnes demonstrated for the first time the carcinogenicity of NDMA. These investigators reported the induction of liver tumors in rats by feeding NDMA. Extensive research on N-nitroso compounds (N-nitrosamines and N-nitrosamides) has revealed that the majority of these compounds are carcinogenic (Swann, 1975). Furthermore, some are mutagenic, teratogenic and embryopathic when administered to pregnant animals. In addition, research in the last fifteen years has also revealed significant information on the occurrence of N-nitrosamines and their formation in different foods and beverages. Progress has also been made on their analysis and extraction from food systems. In 1968, Sander was the first to report N-nitrosamine formation by microorganisms. He demonstrated the ability of four strains of nitrate-reducing enterobacteria to produce N-nitrosamines enzymatically from nitrate and secondary aromatic amines. This study was initiated to further investigate the role of bacteria in the formation of N-nitrosamines from immediate precursors (dimethylamine and nitrite), and to investigate the possibility of N-nitrosamine formation from several other compounds (secondary precursors) via the action of selected bacteria. Studies in various laboratories in recent years have shown that a variety of cured meat products contain trace amounts of N-nitrosopyrrolidine (NPYR) and NDMA (Scanlan, 1975; Crosby and Sawyer, 1976; Sen, 1980; Gray, 1981). The preparation of gid-deed (a delicacy food product made from lamb meat in Libya and most Islamic countries in North Africa ) involves such steps as dry salting of the meat, sun drying and oil frying. Although nitrates or nitrites are not intentionally added to the product, nitrate may be present as an impurity in the salt and accidentally added in the salting step. Through the action of bacteria,nitrate becomes a source of nitrite which may be available for reaction with the secondary amines present in the meat system. Therefore, one further objective of this study was to investigate the possibility of N-nitrosamine formation in fried and non-fried gid-deed samples produced under con- ditions similar to those encountered in the domestic prepa- ration of gid-deed. LITERATURE REVIEW N-Nitrosamines are organic compounds that result from the interaction of amines (primary, secondary or tertiary amines) with nitrite. They have been known since the nine- teenth century; however, it was not until quite recently that they became the subject of considerable interest and controversy. This renewed interest was generated by the work of Magee and Barnes (1956) who demonstrated the carcinogencity of dimethylnitrosamine (NDMA) when fed to rats. Since then, many workers have investigated the biological effects of N-nitrosamines and the majority of these compounds have been shown to be carcinogenic (Preussmann ££._l-9 1976). Furthermore, some are mutagenic, teratogenic and embryopathic when administered to pregnant animals (Montesano and Bartsch, 1976). N-Nitrosamines are characteristically yellow, orange- yellow liquids or solids at room temperature (Scanlan, 1975). They have the common chemical formula: N-N / R II C where R is an alkyl group and R1 is an alkyl, aryl or a wide variety of functional groups (Gray gt 11., 1979). NDMA, N-nitrosodiethylamine (NDEA) and N-nitrosopyrrolidine (NPYR) are among the classical examples isolated from food systems (Sen, 1972; Fazio 33 al., 1973; Fong and Chan, 1973; Eisen- brand gt 31,, 1977). NDMA is a symmetrical dialkylnitrosa- mine while NPYR is a heterocyclic N-nitrosamine. Precursors of N-Nitrosamines and the Nitrosation Reaction As stated by Gray _t al. (1979), the principal mode of N-nitrosamine formation in foods is the reaction between secondary amines and nitrous acid. Nhen secondary amines are treated with nitrous acid, N-nitrosation proceeds and a N-H + HN02——-> N-N=0 + H o / R R 2 stable N-nitrosamine is formed as shown in the equation above. Quaternary ammonium compounds and tertiary amines can also react with nitrous acid to form N-nitrosamines; however, the yields are much lower compared to secondary amines (Fiddler 3; 31., 1972). When a primary aliphatic amine is reacted with nitrous acid, an unstable N-nitrosamine is formed which tautomerizes to diazohydroxide. Hydrolysis of this intermediate produces an unstable diazonium ion. The latter loses nitrogen to form a carbonium ion which can undergo additional elimination and rearrangement reactions (Scanlan, 1975). Primary aliphatic amines can also undergo N-nitrosation reactions to produce stable N-nitrosamines (Narthesen gt gl., 1975). However, the N-nitrosation reac— tion apparently requires the conversion of the primary amines to secondary amines before the N-nitrosation step. The N-nitrosation reactions of various secondary amines and amino acids by nitrous acid have been extensively studied by Mirvish (1972, 1975). These studies indicated that the actual N-nitrosating species can be one of the following depending on the N-nitrosation conditions: nitrous anhydride (N203), nitrous acidium ion (N0+), nitrosyl halide (NOX) or nitrosyl thiocyanate (NOCNS). The rate of the N-nitrosation reaction is affected by the concentration of nitrite and the secondary amine, the basicity of the amine and the pH of the system (Mirvish, 1970). It is also affected by tempera- ture and the presence or absence of catalysts and inhibitors (Mirvish, 1970; Mirvish, 1972; Foreman and Goodhead, 1975). In general, the rate of formation of N-nitrosamine from nitrite and secondary amine follows first order kinetics with respect to the amine concentration, and second order kinetics with respect to nitrite concentration, i.e., the rate of reaction doubles with doubling the concentration of the amine and increases four times with doubling the nitrite concentration (Mirvish, 1970). The rate of formation of N-nitrosamines from secondary amines can be calculated from one of the following equations: Rate = K x (unprotonated amine concentration) (nitrous acid) ........................ (1) Rate = x (total amine concentration) KITTthefl ............................. (2) However, it Should be pointed out that K in equation (1) is independent of pH, while K1 in equation (2) is pH dependent. Under standard conditions of temperature and total Species concentration, Mirvish (1970) showed that with dimethylamine and nitrite, maximum NDMA formation was obtained at pH 3.4. The percent yield decreased on both sides of this optimum pH. He also reported that over a pH range of 9 to 5, the rate of N-nitrosation increased 10 fold for every unit drop in pH. The basicity of the amine has also been shown to affect the rate of N-nitrosation reactions. Mirvish (1971) showed that at constant pH around 3, the relative N-nitrosation rate of the total species of amines increased 1.85 x 105 times on preceeding from piperidine (pr 2.8) to piperazine (pr 8.4) (Table 1). The non-ionized species seemed to have similar reactivities and the rate increased only 4 times for the same amines at pH 3.0. This indicated that the Table 1. Optimum pH and rate constants for the N-nitrosation of some secondary amines and amino acids.a Amine pKa Optimum pH Rate Constant Piperidine 11.2 3.0 0.027 Dimethylamine 10.73 3.4 0.10 Morpholine 8.7 3.0 14.8 Mononitrosopiperazine 6.8 3.0 400.0 Piperazine 5.57 3.0 5000.0 L-Proline -- 2.25 2.9 L-Hydroxyproline -- 2.25 23.0 Sarcosine -- 2.5 13.6 aAdapted from Mirvish (1972). N-nitrosation reaction rate is inversely proportional to the basicity of the amine involved. The rate of N-nitrosation of amino acids was investi- gated by Mirvish (1971) who showed that the Optimum pH for the reaction is between pH 2.2 and 2.5 (Table 1). Again, only the non-protonated amines in the amino acid molecule were able to undergo N-nitrosation reactions, and sarcosine and hydroxyproline are N-nitrosated much more rapidly than dimethylamine and proline. The complicated ionization of these amino acids has been suggested to be the cause for the observed shift in optimum pH for the N-nitrosation reaction, as well as the reaction rate compared to that of secondary amines (Mirvish, 1972). The effect of temperature on N-nitrosation reactions has also been studied. HAS in other chemical reactions, the rate of N-nitrosation is doubled for every 10°C (18°F) rise in temperature (Foreman and Goodhead, 1975). Prolonged storage conditions have also been shown to influence N-nitrosamine formation even at low temperature. Ender gt _1. (1967) studied the effect of prolonged storage of N-nitrosamine formation from dimethylamine and nitrite at pH 6.3 and 4°C. The concentration of each reactant was 40 mM. Their data indicated that 1.6 and 133.5 mg/kg of NDMA were formed when the solution were stored for 2 days and 157 days, respectively. Catalysis and Inhibition of N-Nitrosation Reactions The presence of weak anions such as bromide, chloride and iodide as well as acetate and thiocyanate in the reaction mixture has been shown to accelerate the N-nitrosation reaction (Boyland gt _l., 1971; Boyland, 1972; Fan and Tannenbaum, 1973). Keefer and Roller (1973) studied the effect of carbonyl compounds on N-nitrosamine formation. Their results indicated that the N-nitrosation of secondary amines such as dimethyl- and diethylamine and pyrrolidine was actually catalyzed by the presence of formaldehyde and other carbonyl compounds even under alkaline conditions. It was suggested that catalysis occurred as a result of a reaction between the aldehyde and the secondary amine which l Ln". m 1! 5|. 1‘u 10 gave rise to an intermediate iminium ion. The latter is directly attacked by the nitrite ion and the resulting complex decomposes to N-nitrosamine and the catalytic alde- hyde. However, tertiary and quarternary amines are not affected by such catalysis (Roller and Keefer, 1974). The role of microorganisms in catalyzing N-nitrosation reactions and N-nitrosamine formation have been studied by many workers (Sander, 1968; Ayanaba and Alexander, 1973; Maduagwu and Bassir, 1979). A detailed discussion of the role of microorganisms of N-nitrosamine formation will be presented elsewhere in this review. Similarly, the inhibitory effect of certain chemicals when present in the N-nitrosation system has been widely studied. Fiddler gt _1. (1973) showed that ascorbate and isoascorbate can inhibit NDMA formation in frankfurter cures and in frankfurters. It was also found that the N-nitrosa- tion of morpholine was inhibited by ascorbate (Mirvish 1., 1972) and was completely inhibited when the ratio of e_t_ ascorbic acid to nitrite was greater than two at pH 4.0 and 25°C (Fan and Tannenbaum, 1973). Other compounds such as a-tocopherol, glutathione, cysteine, propyl gallate and other antioxidants have been shown to inhibit N-nitrosamine formation in foods as well as in vivo (Gray and Dugan, 1975; Mirvish, 1975; Sen gt gl., 1976). It has been suggested that these N-nitrosation inhibitors perform their function by competing for available nitrite in the reaction t) We me an 11 mixture, thereby reducing the effective nitrite concentration and consequently reducing the reaction rate (Mirvish, 1970; Sen, 1980). Catalysis of N-Nitrosamine Formation by Microorganisms Sander (1968) was the first to report N-nitrosamine formation by microorganisms. He demonstrated the ability of four strains of nitrate-reducing enterobacteria to produce N-nitrosamines enzymatically from nitrate and secondary aromatic amines at neutral pH values. Hawksworth and Hill (1971) confirmed the results of Sander (1968) using a number of strains of Escherichia coli isolated from the human intestinal tract. They also showed that some nonnitrate-reducing strains of lactobacilli, group D strepto- cocci, clostridia, bacteroids and bifidobacteria can N-nitro- sate secondary amines with nitrite at neutral pH. They have also demonstrated that out of ten strains of E. ggli tested, five were able to form N-nitrosamine when incubated aerobi- cally in a nutrient broth medium containing nitrate and the secondary amines, dimethylamine, diethylamine, diphenylamine, piperidine, pyrrolidine and N-methylaniline. They reported that N-nitrosation was actually observed, even when glucose was omitted from the incubation mixture and the pH of the medium did not fall below 6.5. They concluded that the N-nitrosation could not be due to acid catalysis (Hawksworth and Hill, 1971). 12 Klubes _t _t. (1972) studied the factors which affect N-nitrosamine formation from secondary amines and nitrates by rat intestinal bacteria. They showed that N-nitrosation of (140}dimethylamine by rat intestinal bacteria was suppressed to a certain degree by boiling the bacterial preparation, by omitting glucose, or adding neomycin to the standard incuba— tion system. On the other hand, Klubes _t g1, (1972) found that N-nitrosation of the secondary amine by bacteria was enhanced by the inclusion of riboflavin in the incubation medium. Like t. ggtt, streptococci have been also implicated in N-nitrosamine formation from secondary amines and nitrites. Collins-Thompson gt _1. (1972) demonstrated that formation of NDMA and NDEA from dimethylamine and diethylamine, respec- tively, and nitrite by Streptococcus faecalis, t. faecium and S. lactis. However, their results indicated that N-nitrosa- mine formation by streptococci was non-enzymatic, and the growing and autoclaved cultures or the millipore-filtered culture medium produced equal amounts of N-nitrosamines which were three times greater than the blanks. Their results with 5. £211 were negative for N-nitrosamine formation. N-Nitrosa- mine formation by proteus species from dimethylamine and nitrite has also been documented (Thacker and Brooks, 1974). They showed that NDMA was produced by Protegg morganii, E. ggttgeri and 3. mirabilis but not by three strains of P. vulgaris when grown under the same conditions. be 13 Contrary to the findings of Collins-Thompson gt gt. (1972) who Showed that Staghylococcus aureus was incapable of N-nitrosating dimethylamine, Ishiwata gt gt. (1976) demonstrated that both S. aureus and t. egidermidis produced appreciable quantities of NDMA when incubated in a culture medium containing brain heart infusion, glucose, dimethyl- amine-HCl and nitrate. The inclusion of glucose in the medium enhanced the N-nitrosation reaction, but did not change the pH when a short term incubation (2 hr) was used. Resting cells of g. coli, Cryptococcus terreus, Xanthomonas compestris and Pseudomonas stutzeri have been shown to produce NDMA from nitrite and dimethylamine (Mills and Alexander, 1976). However, only 3. stutzeri was capable of forming NDMA during growth. The acceleratory role of Bacillus species on N-nitro- samine formation was studied by Yang gt g1. (1977). Their results indicated a 12-49 fold enhancement over the initial rate when g. brevis was incubated with dihexylamine and nitrite at pH 3.5. The increase in the N-nitrosation reac- tion rate was similar for both boiled and untreated cells. Molds have also been shown to be active in N-nitrosamine formation. Ayanaba and Alexander (1973) reported that NDMA appeared in cell suspensions of E. coli, Strggtococcus epi- dermidis and in the hyphal mats of Aspergillus oryzae incu- bated in a solution containing dimethylamine and nitrate. l4 Yeast, as well, have been shown to catalyze N-nitrosa- mine formation from immediate precursors. Maduagwa and Bassir (1979) reported that yeast A (a wild yeast isolated from fermenting palm wine) but not Saccharomyces, was able to form N-nitrosamine from dimethylamine and nitrite. The yeast failed to N-nitrosate the secondary amine when nitrite was replaced by nitrate. Similarly, no N-nitrosamine was detected when trimethylamine was used in place of dimethyla- mine. In another study, Yang gt g1. (1977) showed that Saccharomyces cerevisia and Saccharomycggsis lipolytica were able to N-nitrosate a group of secondary amines including dimethylamine, diethylamine, dibutylamine, dihexylamine, morpholine and piperidine. The highest rate of N-nitrosation was obtained with dihexylamine when the reaction mixture was adjusted to pH 3.5. They also showed that at a 5 mg/ml cell concentration of g. ligotytica, the rate of N-nitrosation increased 80 fold over the N-nitrosation rate in the blank. Resting cells of Q. terreus also N-nitrosated dimethylamine. The growing cell suspension as well as boiled cell suspen- sions did not enhance N-nitrosamine formation (Mills and Alexander, 1976). Formation of NDMA from dimethylamine and nitrate by microorganisms used in the baking industry or isolated from raw materials of bakery products was studied by Iubu and Bogovski (1978). They showed that two Saccharomyces cerevisiae strains used in the baking industry, two 15 Pseudomonas herbicola strains isolated from cereals, one Pseudomonous species isolated from rye, a strain of Bacillus subtilis isolated from leaven and another Pseudomonous strain isolated from compressed bakers yeast, a11 produced NDMA when incubated in a chemically defined medium con- taining dimethylamine and nitrate. Role of Microorganisms in N-Nitrosamine Formation In general, most of the past research involving micro- bial formation of N—nitrosamines can be classified into three categories: 1. The first deals with the formation of N-nitrosamines from immediate precursors (nitrite and secondary amines). 2. The second relates to organisms capable of either reducing nitrate to nitrite, or metabolizing certain substrates to secondary amines which can react with nitrite to form N-nitrosamines. 3. The last category deals with organisms capable of converting N-nitrosated precursors to N-nitrosamine as their final product. As far as the first category is concerned, the catalytic role of microorganisms in N-nitrosamine formation has been confirmed beyond any reasonable doubt. However, there are two schools of thought in regard to the catalytic nature. Klubes gt g1. (1972), Ayanaba and Alexander (1973) and ff— ot or th be Mic 16 Kunisaki and Hayashi (1979) have concluded that N-nitrosa- mine formation by microorganisms is enzymatic in nature. They showed that boiled cell suspensions of different organisms were unable to N-nitrosate dimethylamine in the presence of nitrite, while living cells or their extracts did. 0n the other hand, Collins-Thompson gt gt. (1972) and Yang gt gt. (1977) demonstrated that boiled cells were equally active in N-nitrosating secondary amines in the presence of nitrite, and concluded that the N-nitrosation reaction is non-enzymatic. They hypothesized that other cell constituents, but not enzymes, were responsible for N-nitrosation catalysis. Collins-Thompsongt g1. (1972) suggested that one or more of the metabolic products produced by Streptococci were responsible for catalyzing N-nitrosamine formation, while Yang gt gt. (1977) proposed that the accele- ration of the N-nitrosation reaction in the presence of bacterial cells was caused by the hydrOphobic interaction of the precursor amine with cellular constituents, possibly a component of the cell wall, the cytoplasmic membrane or other ultracellular membranous structure. Production of organic acids by microorganisms lowers the pH and favors the chemical N-nitrosation reactions (Sander and Schweins- berg, 1972). Reduction of nitrates to nitrites by microorganisms is a classical phenomenon in microbiology, and a wide range of microorganisms including certain species of yeasts, molds 17 and bacteria can convert nitrates to nitrites. Dimethylamine has been shown to be produced by several bacterial species when incubated with trimethylamine. Ayanaba gt g1. (1973) reported that microorganisms in sewage converted trimethylamine to dimethylamine with subsequent formation of NDMA when the culture medium was amended with trimethylamine and nitrite. Concurrently, Ayanaba and Alexander (1973) stated that the mold, Mortierella_ggrvis- Egtg and an unidentified bacterium converted trimethylamine to dimethylamine, and only the bacterium produced NDMA in the presence of nitrite. Serratia, a soil bacteria, was also shown to be active in the conversion of trimethylamine to dimethylamine, and ultimately used in N-nitrosamine formation in the presence of nitrite (Maduagwu and Bassir, 1979). Amino acids and N-nitrosamino acids have been shown to be decarboxylated by bacteria producing secondary amines and N-nitrosamines. Kawabata and Miyakoshi (1976) showed that out of 30 organisms tested, 7 were able to decarboxylate L-proline to pyrrolidine. These included two strains of E. coli, Bacillus cereus var. mycoides ATCC 1178, B. maga- terium IAM 1166, Bacillus circulans, t. aureus and Flavobac- terium xerosis IFO 3752. However, only two Pseudomonas species, fig. fluorescegg AHU 1143 and fig. schulkilliensis were able to decarboxylate N-nitrOSOproline to NPYR giving yields of 0.3 to 0.02% respectively. They also stated that 18 no close relationship was observed in the distribution pattern of L-proline and N-nitrosoproline decarboxylases among the test organisms, i.e., none of the organisms that decarboxylated L-proline were able to decarboxylate N- nitrosoproline and vice versa. N-Nitrosamine Degradation by Microorganismg Very few reports have been published regarding N- nitrosamine degradation by microorganisms. Klubes gt_gl. (1972) stated that preformed (14C) NDMA incubated with cecal bacteria of the rat under standard conditions for 20 hours appears to be stable and was recoverable in 95% yield from incubation mixtures. In a similar study, Rowland and Grasso (1975) reported that a major proportion of bacterial types commonly found in the gastro-intestinal tract of many animals and man were active in degrading N-nitrosodiphenylamine and NDMA, the former being degraded more rapidly than the latter. In this study, approximately 55% of added N-nitrosodiphenyla- mine, 30% of NPYR, and 4% of NDMA were degraded when the concentrations of the N-nitrosamines were less than 0.05 umol/ml at pH 7.0 per 20 hour incubation. E. coli, Bacteroides, Bifidobacterium, Lactobacillus and S. faecalis were among the organisms tested, along with several potential pathogens; Klebsiella _grogenes, E. aeruginosa and Proteus species all were found to be active in N-nitrosamine degra- dation. Recently, Harada (1980) studied the effect of the Ir“. Hi vi if m“ EV of are [1165‘ 19 conditions of growth and enzymic reaction on the decomposi- tion of N-nitrosamines by Rhizopus oryzae. He showed that a pH of 8.0 and a concentration of 0.03 - 0.12 mmole of N-nitro- samine at 20°C were optimal for NDMA degradation. However, N-nitrosodiphenylamine breakdown was optimal at 30°C and 0.1 mmole concentration. The cell-free enzyme system was also active in degrading N-nitrosamines. The degradation activities were highest at pH 8.0 and 30°C and at N-nitrosa- mine concentrations greater than 0.1 mmole. Occurrence of N-Nitrosamines in Foods AS stated by Crosby and Sawyer (1976), the first indi- cation of the presence of N-nitrosamines in the environment came from studies of a disease of mink in Norway which was characterized by extensive liver damage. In 1957, the injurious effects were first observed and the toxic factor was later isolated and identified as NDMA (Ender gt gl., 1964). The carrier was herring meal which has been processed with high levels of nitrite and fed to the animals. Since then, several N-nitrosamines have been shown to occur in a wide variety of food products and alcoholic beverages. How- ever, NDMA and NPYR are, by far, the most frequently detected N-nitrosamines in food products. N-Nitrosamines in Cured Meats. As far as the occurrence of N-nitrosamines in foods is concerned, cured meat products are the products of major significance (Gray, 1981). Cured meats such as bacon (cooked or fried) and to a much lesser 20 extent, ham and certain cured sausage products including dry sausages, uncooked salami and frankfurters, are the major contributors of N-nitrosamines in the American diet (Sen, 1972; Fazio _t _l., 1973; Gray gt _l., 1977; Sen, 1980). A summary of reports on the occurrence of N-nitrosamines in cured meat products is presented in Table 2. Among all cured meat products tested, cooked bacon has consistently been Shown to contain relatively high levels of N-nitrosamines, mainly NPYR and traces of NDMA (Sen, 1980). Since NPYR has not been detected in raw bacon, NPYR is believed to be formed during the high cooking temperatures utilized in the frying process (Sen, 1980; Gray, 1981). Although several compounds have been suggested as possible precursors for NPYR in cooked bacon (putrescine, collagen, spermidine, pyrrolidine), free proline which is present in concentrations of approximately 20 mg/kg in pork belly, is believed to be the major precursor (Gray, 1976; Lakritz gt gl., 1976; Gray and Collins, 1977; Bharucha _t _l., 1979). Model system studies have indicated that N-nitrosation of proline followed by decarboxylation is the major pathway by which NPYR is formed in cooked bacon as opposed to decarboxylation of proline to pyrrolidine and subsequent N-nitrosation to NPYR (Bharucha gt gl., 1979; Lee, 1981). The level of NPYR in the cooked product depends upon well documented factors (Gray, 1981). These include nitrite 21 Table 2. N-Nitrosamines detected in various cured meat products Meat product N-nitrosamine (pg/kg) Investigator Fried bacon NPYR trace-40 Crosby _t g1: (1972) Cooked-out fat Fried bacon NPYR 4-25 Sen gt g1, (1972) Cooked-out fat Fried bacon NDMA, NPYR trace 44 Sen gt_gl, (1973) Cooked-out fat Fried bacon NPYR 4-100 Havery _j;gl, (1973) Cooked-out fat Fried bacon NPYR trace-41 Gray gt gt. (1977) Cooked-out fat Fried bacon NPYR, NDMA 2-34 Sen gt _1. (1979) Cooked-out fat Fried bacon NPYR 2-34 Pensabene gta__l_: (1980) Cooked-out fat Frankfurters NDMA 11-84 Nasserman. gt_a_1_. (1972) Dry sausage NDMA 20-80 Sen (1972) uncooked salami Smoked horse meat NDMA, NDEA trace-25 Groenen gt_ l.(l976) 22 concentration, frying temperature and time, and the presence or absence of N-nitrosamine inhibitors such as ascorbates and lipophilic inhibitors. N-Nitrosamines in Fish and Fish Products. Fish, fish meal and other seafoods have also been shown to contain appreciable amounts of NDMA, and to a lesser extent NPYR and NDEA (Table 3). As an example, Fong and Chan (1973) Table 3. N-Nitrosamines reported in fish and other seafoods. Fish Product N-nitrosamine (pg/kg) Investigator Herring meal NDMA (15,000-1000,000) Ender _t _a_1. (1964) (nitrite Sakshaug gt__l. (1965) treated) Smoked fish NDMA, trace-40 Ender and Ceh (1967) Smoked fish, NDMA, 4-26 Fazio gt _1. (1971) nitrite or nitrate treated Salted and dried NDMA, 10-1000 Fong and Chan (1973) fish Various Chinese NDMA, NPYR, trace-37 Fong and Chan (1977) seafoods have detected fairly high levels (10-1000 ug/kg) of NDMA in salted and dried marine fish. The presence of nitrate (as an impurity) in crude salt appears to be instrumental in N-nitrosamine formation. Hhen crude salt was replaced by refined salt, and benzoic acid was added to control 23 microbial growth, apparently only small amounts of N- nitrosamine were detected (Fong and Chan, 1976). Herring meal, treated with nitrite has been found to contain as high as 100 mg/kg of NDMA (Sakshaug gt _l., 1965). Sig- nificantly lower levels (450 ug/kg) were detected in untreated fish meal (Sen gt gl., 1972). The presence of NDMA in salted and/or cured fish products is not unexpected since dimethylamine, trimethy- lamine and trimethylamine oxide have been reported to be present at fairly high concentrations in fish and fish products (Shewan, 1951; Kawamura gt 1., 1971; Singer and Lijinsky, 1974). N-Nitrosamines in Dairy Products. Trace amounts to several parts per billion of NDMA, NDEA, N-nitrosopiperidine (NPIP) and NPYR have been detected in a variety of cheeses including Cheddar, Gouda, Edam, Camembert and Havarti cheese (Crosby gt 1., 1972; Eisenbrand e 1., 1977; Sen t 1., 1977). Table 4 lists some of the dairy products from which N-nitrosamines have been isolated. N-Nitrosamines in Other Food Products. NDMA has been shown to be present in a variety of alcoholic beverages including beer (Spiegelhalder gt gl., 1979; Havery gt gl., 1981), palm wine (Bassir and Maduagwu, 1978) and Scotch whiskey (Goff and Fine, 1979). NDMA also has been detected in edible oils and margarine. Levels of 0.22-1.01 ug/kg were reported in several commercial oils including corn, Table 4. 24 products N-Nitrosamines (pg/kg) detected in selected dairy Dairy Product N-Nitrosamine and level present Investigator Cheese Havarti and Gouda Cheshire Gouda, Havarti, Camembert, Cheddar Nine cheese Yogurt Dried buttermilk Nonfat dried milk NDPAa, NDIPA°, 5-10 NDEA, NPYR, T NDMA, NDEA, NPIP, trace-19 NDMA, NDEA, 7-68 NPIP Not detected NDMA 0.9-1.8 NDMA trace-4.5 Kroeller (1967) Alliston gt gt. Sen gt (1972) gt. (1977) _1 (1977) __._l (1977) _£._l (1980) e l (1980) aNDPA, N-nitrosodipropylamine b NDIPA, Nitrosodiisopropylamine 25 olive, sunflower and soybean oils and margarine (Fiddler _t _t., 1981). However, higher levels (up to 23 and 28 ug/kg for NDMA and NDEA, respectively) had been reported earlier by Hedler gt _t. (1979). Commercial curing premixes con- taining nitrates, nitrites, and spices such as black pepper, paprika and salt have also been shown to contain exces- sively high levels of N-nitrosamines (850-25,000 ug/kg). Sen gt gt. (1973) found that when nitrite was mixed with paprika, NPYR was formed, whereas nitrite plus black pepper resulted in NPIP formation. Similar results were reported later by Gough and Goodhead (1975). Precursors and N-Nitrosamine Formation in Foods In foods, N-nitrosamines are formed principally by the same chemical or biochemical reactions discussed earlier, where naturally occurring amines react with nitrites added to the food system or produced by bacterial reduction of naturally occurring nitrates (Gray, 1981). The N-nitrosation reactions are also affected by the same factors: pH, concen- tration of nitrite and secondary amine, basicity of the amine being N-nitrosated and the presence or absence of inhibitors and catalysts. Amines in Foods. a) Fish and fish products. Historically, amines have been associated with fish products (Maga, 1978). Table 5 summarizes some of the amines reported to be present 26 Table 5. Some of the amine compounds reported to be present in fish and fish products. Fish product Amine (#3::g) Investigators Raw sardine Dimethylamine 5.9 Kawamura gt__t, (197” Roasted sardine " 48.6 " Oiled sardine " 179.6 " Raw pollack roes " 116.6 " Roasted pollack " 205.7 " roes Flaked tuna " 12.6 " Oiled tuna " 13.1 " Canned tuna " 23.2 Singerand Lijinsky(l976) Perch " 180 " Bass " llO " Cod " 738 " Trout " 7 " Raw crab " 0.3 Kawamura gt gt, (1971) Raw shrimp " 4.0 " Raw oyster " 0.1 " Fish protein " 151.0 Miller gt_gt. (1973) concentrate Fish Trimethylaminel400 " Fish protein " 5.1 " Trout Dipropylamine 0.4 Singer andljjinsky(1976) 27 in some fish products. Trimethylamine, trimethylamine oxide and dimethylamine are found at fairly high concentrations in various fish, particularly those of marine origin. Concentrations as high as 100-185 mg/100 g of fish have been reported (Shewan, 1951). Several other amines such as methylamine, propylamine, butylamine and pentylamine along with tyramine, putrescine and cadaverine have been reported to be present in some fish products (Basco and Barrera, 1972; Cantoni gt al., 1974; Golovnya _t _t., 1967; Minakowski and Mathias, 1967). As stated by Koprowski (1968), Dyer (1952) cited three sources of trimethylamine and/or trimethylamine oxide in fish. First, it may be present in the food supply. It has been reported that marine algae and plants as well as zoo- plankton contain more of these amines than do the comparable fresh water species (Tarr, 1941; Groninger, 1959). Secondly, trimethylamine and trimethylamine oxide can be formed by bacterial degradation of choline and choline-containing substances such as phosphorylcholine and lecithin (Dyer and Hood, 1947; Bilinski, 1962). Aerobacter aerggenes, E. rettgeri, 3. ichthyosmigg, E. mirabilis and Schigella alkalescens have been shown to be capable of decomposing choline to trimethylamine (Dyer and Hood, 1947). The third mechanism which has been suggested was de novo synthesis of trimethylamine from ammonia through a series of methylations on the ammonia molecule to form methylamine, dimethylamine 28 and then trimethylamine (Dyer, 1952). Trimethylamine can be oxidized in fish liver to trimethylamine oxide as proposed by the same investigator. Trimethylamine oxide is present at higher concentrations in marine fish than in fresh-water fishes because it is necessary for osmotic pressure regula- tion in marine fish, while it is not necessary for that purpose in fresh-water fish and is simply excreted in the urine (IJyer, 1952). Trimethylamine levels have been used to judge the freshness of fish, as influenced by storage. It has been reported that good quality sable fish contains 147 mg trimethylamine/kg of fish whereas the level in spoiled sable fish was in excess of 1100 mg/kg (Gruger, 1972). b) Meats and meat Eroducts. As has been mentioned previously amines are naturally present in meats and meat products. Dimethylamine, diethylamine and dipropylamine, as well as spermine, spermidine and tyramine, have been iden- tified in a variety of meat products (Spinelli gt gt., 1974; Lakritz gt__t., 1975; Singer and Lijinsky, 1976). Gray and Collins (1977) determined the free proline concentration, a possible precursor of NPYR (Bills gt gt., 1973; Gray and Dugan, 1975) Tn five green pork bellies stored at 2°C for 1, 8, 15 and 28 days. Their results indicated an average of 11.8 OM free proline/100 g of whole tissue sample (lean and adipose). Storage of samples at 2°C for 28 days resulted in an approximate three-fold increase in proline content in both whole belly and lean tissue and a four-fold increase 29 in the adipose tissue. Similarly, 18.3 to 31.6 umoles of free proline were Tound to be present in 100 9 tissue for five commercial bacon samples (Gray gt _t., 1977). The effect of heating and putrefaction on pork amine levels have been studied by Lakritz gt gt. (1975). They reported that putrefaction dramatically increased the levels of spermine, spermidine and putrescine in pork, while cooking reduced them slightly. Similar trends were observed with the amines, cadaverine, histamine, tryptamine and tyramine. Lecithin, phosphorylcholine and choline (amine containing compounds), which have been proposed as possible precursors for NDMA (Pensabene gt gt., 1975; Gray _t gt., 1978), occur naturally in meats. The lecithin content of fresh hog belly was quan- titated by Kuchmak and Dugan (1963) who reported levels of 0.34 g per 100 g of fresh hog belly. Sarcosine, which can be N-nitrosated much more rapidly than dimethylamine, occurs widely in nature as a result of protein degradation. Degra- dation of proteins under anaerobic conditions by microorga- nisms may significantly increase the free amine content of histamine, tyramine, piperidine, putrescine and cadaverine (Frazier, 1967). c) Dairy products. Several secondary amines, dimethylamine, diethylamine, dipropylamine, dibutylamine, pyrrolidine and methylbutylamine have been isolated and identified in a number of dairy products including evaporated whole milk, low fat yogurt and a wide variety of cheeses 30 Golovnya and Mironov, 1968; Ney and Hirotama, 1971; Singer and Lijinsky, 1976). Trimethylamine, triethylamine and tripropylamine have been reported to be present in Russian cheese (Golovnya and Mironov, 1968; Golovnya and Zhurav- leva, 1970). De Vuayst gt _t. (1976) analyzed several cheeses for histamine, tyramine, putrescine and cadaverine and reported that blue cheese had the highest total amine content, and the highest histamine level (4.093 mg/g cheese). However, cheddar contained more of the other amines than all the other cheeses analyzed. d) Amines in other foods and alcoholic and non- alcoholic beverages. As reviewed by Maga (1978), primarily volatile amines are present in beer. Dimethylamine, pyrroli- dine and dimethylbutylamine have been reported to be present at relatively low concentrations (0.65 mg/kg, trace, 0.16 mg/ kg, respectively), with ethylamine being present in the highest concentration among the volatiles (1.04 mg/kg (Palmand _t gt., 1971; Slaughter gt gt., 1971; Singer and Lijinsky, l976). Dimethylamine and trimethylamine were also reported to be present in cocoa and tea leaves, and in coffee along with other polyamines (Serekov and Proiser, 1960; Amorim t al., 1977; Marion t 1., 1967). Various spices, such as paprika and black pepper, contain high levels of amines including pyrrolidine and piperidine (Gough and Goodhead, 1975). 31 e) Biologically active amines in foods. Biologi- cally active amines are normal constituents of many foods, but they usually do not represent any hazard to individuals unless large amounts are ingested, or the natural mechanism for one or more of the amines is inhibited or genetically deficient (Rice _t _t., 1976). They are defined as alipha- tic, alicyclic or heterocyclic organic bases of low molecular weight which are metabolically produced in plants, animals and microorganisms (Franzen and Eyesell, 1969). These com- pounds include phenethylamine, dopamine, norepinephrine and tyramine (vasoactive pressor amines which increase blood pressure when ingested) and serotonin and histamine, strongly vasoactive amines. Histamine is a strong capillary dilator and can produce hypotensive effects i.e., reduce blood pressure (Goodman and Gilman, 1965). Biologically active amines have been detected in many foods including cheese, fish products, meat extract, sauer- kraut, wines and yeast extract (Blackwell and Mabbitt, 1969; Sen, 1969; Ienisten, 1973; Mayer and Pause, 1972). As an example, tuna fish has been reported to contain as high as 5000 pg histamine/g while the histamine content of cheese varies from 0 to 2600 pg/g (Ienisten, 1973; Voigt et al., 1974), depending on the type of cheese and on how long it is aged. Generally speaking, fresh foods contain small amounts of tyramine and histamine; however, fermentation and/or 32 spoilage of these products via microbial degradation of amino acids increase their amine content (Rice _t., 1976). It gt has been reported that several strains of g. faecalis along with t. ggorogenes, t. aerofoetidum, E. coli, 3. mirabilis and fig. reptilivora possess tyrosine decarboxylase activity and can product tyramine from tyrosine (Gale, 1940; Mossell, 1968). Histidine decarboxylase activity have also been detected in four Lactobacillus species including Lacto- bacillus 30a (Rodwell, 1953). Nitrates and Nitrites in Foods Nitrates are natural components of the environment and constitute the primary source of fixed nitrogen for green plants (Gray and Randall, 1979). They occur in relatively high concentrations in vegetables such as lettuce, cabbage, cauliflower, spinach, celery, carrots, beets and radishes. Sometimes values as high as 1000-3000 mg/kg have been repor- ted (Nhite, 1975). Nitrates also occur in drinking water, and when their concentration exceeds 20 mg/kg, water becomes the major source of nitrate dietary intake (Hawksworth and Hill, 1971). Nitrates also enter our foods as impurities in crude salt and as intentional food additives in some meat curing premixes. Nitrates were added along with nitrites in cure preparations for ham, bacon, frankfurters, cured fish and corned beef. They were added to act as a reserve for nitrites. Nitrates are also added to cheese milk in some European countries to inhibit the growth of Clostridium 33 tyrobutyricum spores which may survive the normal pasteuri- zation process (Goodhead gt gt., 1976). It was estimated that out of the 99.3 mg average daily consumption of nitrates in the U.S., only 9.4% comes from cured meat, and a large proportion, 86.3%, comes from vegetables (White, 1976). Nitrites are present at lower concentrations in our diet than are nitrates. However, the latter can readily be re- duced to nitrite by a variety of microorganisms, such as t. ggtt, various salmonella and staphylcocci (Bolotou gt gt., 1972) or in vivo in the human body (Tannenbaum gt _t., 1976). Nitrites are added to meats as curing ingredients to inhibit the outgrowth of Clostridium botulinum spores. These orga- nisms produce a very powerful neurotoxin, which may cause death if ingested even at minute quantities (Frazier, 1967; Sofos gt _t., 1979). Nitrites are also added in the curing of meat products to inhibit oxidative rancidity and warmed over flavor (HOF), to fix the desirable pink color, to modify the flavor and improve the texture of cured meat products (Lechowich gt gt., 1978; Sofos gt gt., 1979). It was origi- nally thought that cured meats accounted for approximately 21.2% of the estimated average daily ingestion for U.S. resi- dents (Nhite, 1976). Other sources included fruits, vege- tables, bread and saliva. The latter accounted for 76.8% of the average daily ingestion for U.S. residents. More recent estimates, however, indicate that only 2% of the human expo- sure to nitrite in the United States is a consequence of 34 consumption of meats cured with nitrite (CAST, 1978). The remaining 98% of the exposure is due to other sources, which appear to be almost exclusively dietary nitrogenous substances other than nitrite that undergo transformation in the diges- tive tract with the production of some nitrite (Tannenbaum t 1., 1974; Tannenbaum gt gt., 1978). In Vivo N-Nitrosamine Formation In view of the conditions under which N-nitrosamines are formed, and the fact that N-nitrosation reactions proceed at a faster rate at relatively low pH values close to those of the human stomach, researchers have become interested in examining the possibility that trace quantities of N-nitroso compounds may be formed under the acidic conditions of the human stomach. Studies on concurrent administration of nitrite and various secondary amines to experimental animals have shown that N-nitrosamine formation can indeed occur in the gastro-intestinal tract. Hashimoto gt _t. (1976) detected NDMA in the contents of the stomach, intestine and cecum of rats fed a diet supplemented with 0.1% dimethy- lamine and 0.4% potassium nitrate using 5 strains of NDMA forming bacteria. NDMA was not detected in the control group (their diet did not contain bacteria). As reviewed by Sen (1980), eSOphageal and liVer tumors deveIOped in experi- mental rats when their diets included nitrites and secondary amines (Sander and Burkle, 1969). NPYR was also detected 35 in the feces of two out of three rats tested when they were fed diets containing N-nitrosoproline (Kawabata and Miya- koshi, 1976). Formation of N-nitrosamines from secondary amines and nitrite tg tttgg has also been studied by several workers. Sen gt gt. (1969) demonstrated the formation of NDEA when diethylamine and nitrite were incubated with gastric juices from rats, rabbits, cats, dOgS and man. They also showed that human and rabbit gastric juices (pH 1-2) produced more NDEA than did rat gastric juice (pH 4-5). In vivo N-nitrosation of diethylamine was demonstrated in cats and rabbits. Lane and Bailey (1973) studied the effect of pH on NDMA formation tg tittg in human gastric juice. Their results indicated that over a‘pH range of 1.7-4.5, N-nitrosation of dimethylamine appeared to be optimal at pH 2.5 although NDMA was formed over the entire pH range tested. N-Nitrosamines were also detected in non-normal human gastric contents. Lakritz gt gt. (1978) analyzed the stomach contents (gastric) of fasting patients, hospitalized for a variety of conditions and undergoing routine clinical gastric examinations. Their preliminary data indicated the presence of 5-30 pg NDEA per kilogram in 4 samples, 2 pg NDMA in two samples tested. Dimethylamine, trimethylamine, histamine, cadaverine, putrescine, ethanolamine and trypta- mine were detected (qualitatively) in a pooled sample (Lakritz et al., 1978). 36 Carcinogenicity of N-Nitrosamines Among carcinogens, N-nitrosamines are the most broadly acting and among the most potent. Over 100 N-nitroso compounds have been tested and the vast majority have been found to be carcinogenic (Preussman gt gt., 1976; Lijinsky, 1977; Lijinsky, 1979). Thus far, there is no direct evidence that N-nitrosamines are carcinogenic to man (Gray and Randall, 1979). However, several animal species including sub human primates have been tested and none of these species were able to resist the carcinogenic effect of N-nitrosamines (Sen, 1980). Therefore, it is generally assumed that man is not the exception and these compounds are considered potential hazards to humans unless proven otherwise. According to their mode of action as carcinogens, N- nitroso compounds can be divided into two classes; N-nitro- samides, which act directly, and N-nitrosamines which require enzymatic activation before they exert their carcinogenic effect (Magee, 1971; Lyle _t _t., 1979; Michejda gt gt., 1979). N-Nitrosamines are considered to be among the most versatile carcinogens known to man. They can induce tumors in practically all important organs including liver, kidney, lungs, brain and nervous system, esophagus, stomach, urinary bladder and blood generating organs (Griciute, 1978). Target specificity is governed by the chemical structure of the carcinogenic compound, 37 dosage, mode of administration and duration of exposure. It also depends on animal species and strain (Crosby and Sawyer, 1976; Griciute, 1978). The specificity of certain N-nitrosamines in their ability to attack a specific tissue is explained by the assumption that certain tissues are more capable in metabolizing a specific N-nitrosamine than others. Therefore, liver cancer is more frequent in the animal species tested, because liver is the primary site in which xenobiotics are metabolized (detoxicated or activated). The series of events which take place after N-nitrosa- mines are ingested and which lead to cancer development are unknown. However, many of the mechanisms which have been proposed suggest that methyl alkyl nitrosamines are enzy- matically oxidized and/or hydroxylated on the methyl group to give a-hydroxymethyl N-nitrosamine (Baldwin gt_gt,, 1976; Mechejda gt gt., 1979). This a-hydroxynitrosamine can lose an aldehyde and form the primary alkyl N-nitrosamine R-N-CH3-——-—-—+ R-N-CHZOH N0 N0 which rapidly rearranges and forms an alkyl diazonium ion R-N-CH OH-—-——é R-N=NOH + OCH N0 R-N=NOH -—————9 R-N+ 38 which has the ability to alkylate nucleic acids. s-Hydroxy- lation reactions have also been postulated as a possible mechanism for N-nitrosamines with side chains larger than ethyl. Degradation of these compounds via B-hydroxylation, as suggested, will produce active methylating agents (Kruger, 1973). Whatever the case might be, it has been suggested that the methylating agent alkylates the third position of cytosine or the seventh position of guanine in the DNA and/or the RNA molecule (nucleic acids) in the target tissue and interferes with their normal functions (Shank, 1975). This also explains both the mutagenic and teratogenic activity of these N-nitroso compounds when tested in bacteria and pregnant animals, respectively (Tomatis t 1., 1975; Baldwin _t _t., 1976; Montesano and Bartsch, 1976). The quantitative aspects of exposure in N-nitrosamine carcinogenesis have been studied by several investigators. Magee and Barnes (1956) demonstrated a high incidence of malignant liver tumors in rats fed a diet containing 50 mg/kg NDMA. Kidney tumors were observed when higher doses were used. Preussmann gt gt. (1977) studied the dose response relationship of NPYR in rats and they reported that a statisticaly significant increase in liver cancer occurred when rats were fed daily (in water) 10, 3, 1 mg/kg of body weight. A daily dose of 0.3 mg/kg did not increase significantly the rate of tumor development compared to untreated controls. 39 A similar study with NDMA has shown that all doses tested (2, 5, 10, 20, 50 mg/kg) in the diet of rats caused a significant number of liver tumors, except the lowest dose, which resulted in liver'Ummrs only in one rat. However, no safe dose was established since the untreated controls did not develop liver tumors (Terracine gt gt., 1967). Gid-deed Preparation Gid-deed, a delicacy food product, is prepared tradi- tionally almost in every household in Libya, especially on Aid Al-akbar (a religious day which coincides with the day Ibrahim, the prophet, was asked to sacrifice his son). Animals, preferably yearlings are slaughtered, skinned and eviscerated. Part of the meat is used on the same or the next day and the remaining meat is deboned, heavily salted by rubbing dry salt on meat slices. The salted meat is sun-dried for 2-3 days, depending on the season. The sun-dried product is either stored as is in glazed clay containers at room temperature, or fried in olive oil and stored for use as a delicacy, and used when fresh meat is not available. Originally, the method may have been developed to preserve the meat for later consumption. Today, refrigerators are available for the majority of households but gid-deed is still used in the same manner. 40 Nitrates or nitrites are not intentionally added to the product; however, nitrate may be present as an impurity in the salt and accidentally added in the salting step. This is supported by personal observations of some gid-deed samples which have a bright red color when cooked or after oil frying. No data are available regarding the nitrate content of local salt or N-nitrosamine formation in the final product. EXPERIMENTAL Materials Sarcosine, NDMA and NPYR were obtained from Eastman Organic Chemical Division, Eastman Kodak Co. (Rochester, NY), trimethylamine hydrochloride (TMA-HCl) and trimethylamine- N-oxide dihydrate (TMA-N-O. 2H20) from Aldrich Chemical Company, Inc. (Milwaukee, Wis.), dimethylamine hydrochlo- ride and phosphatidylcholine from Fisher Scientific Company Chemical Manufacturing Division (Fair Lawn, NJ), and choline chloride and phosphorylcholine chloride from Sigma Chemical Company (St. Louis, MO). The microbiological media, including tryptic soy broth (TSB) and brain heart infusion (BHI), were purchased in a dehydrated form from Difco Laboratories (Detroit, Michigan). Other chemicals used were reagent grade and were obtained from commercial suppliers. N-Nitrosoproline and N-nitrososarcosine were synthesized according to the method of Hansen gt gt. (1974). L-proline (hydroxyproline-free) or sarcosine, obtained from Nitri- tional Biochemicals Company (Cleveland, Ohio), was dissolved in deionized distilled water. A ten fold excess of sodium nitrite was added and the pH was adjusted to 3.0 with 41 42 hydrochloric acid. The N-nitrosation reaction was allowed to proceed in the dark, with stirring, for 18 hours at room temperature. The pH was readjusted to 1.0 and the reaction mixture was freeze dired. N-Nitrosoproline was extracted with methylene chloride. The extract was dried over anhy- drous sodium sulfate, filtered, and the methylene chloride evaporated in a rotary evaporator at 40°C. Both N-nitroso- proline and N-nitrososarcosine were stored in dark bottles at -20°C until required. Methods Bacteriology A total of 10 strains of various bacteria consisting of E. coli E-2, E. coli K-12, S. aureus, Microbacterium ther- mosphactum, Lactobacillus bulgaricus, Pseudomonas fluorescens ATCC 13430, Pseudomonas putida biotype A ATCC 15070, Pseudo- monas Bgtida ATCC 27212 and two strains of Pseudomonas schuylkilliensis ATCC 15916 and ATCC 15917 were examined for their ability to N-nitrosate dimethylamine, to decarboxylate N-nitrosoproline (to NPYR) and to produce NDMA from various precursors in the presence of nitrite. All the organisms were grown in a nutrient broth medium (NB) at the optimum temperature for each organism (see later) in screw capped test tubes for 24 hours and then stored under refrigeration at 4°C. Fresh transfers were made in the same medium every 4 weeks. 43 N-Nitrosation of Dimethylamine with Nitrite. Fresh cultures (1 ml), as prepared above, were inoculated into 200 ml of TSB medium containing 0.05% (w/v) NaNO2 and 0.25% (w/v) DMA-HCl (0.1 g and 0.5 g, respectively) in 500 ml Erlenmeyer flasks. Flasks were incubated for 48 hours at the optimum temperatures (37°C for g. ggtt, t. ggt- garicus and g. aureus, and 25°C for the remaining cultures) in a gyratory New Brunswick controlled-environment incubator (Model G-25) set at a speed of 200 rpm. N-Nitrosation of Dimethylamine with Nitrate. Fresh cultures were inoculated into 200 m1 of TSB medium containing 0.25% (w/v) DMA-HCl and 0.25% (w/v) NaN03 in 500 ml Erlen- meyer flasks. The samples were incubated as above. Decarboxylation of N-nitrosoproline and N-nitrososar- cosine. The N-nitrosoproline and N-nitrososarcosine decarboxylase reactions of test organisms were carried out according to Kawabata and Miyakoshi (1976) with minor modifications. Test organisms were inoculated in 100 ml of a medium containing 0.2% (w/v) N-nitrosoproline or N-nitrososarcosine and incubated at their optimum growth temperature with shaking and aeration in a gyratory NBS controlled-environment incubator (Model G-25) at 200 rpm for 48 hours. The resulting NPYR and NDMA (from the decarboxylation of N-nitrosoproline and N-nitrososarcosine respectively) were extracted with methylene chloride and analyzed with a combined gas chromatograph-thermal energy 44 analyzer (GC-TEA) system. NDMA Formation from Other Precursors. The possibility of N-nitrosamine formation via microbial degradation of secondary precursors of NDMA was investigated using the following precursors: trimethylamine hydrochloride (TMA- HCl), trimethylamine oxide-dihydrate (TMA-O: 2H20), choline chloride, phosphorylcholine chloride, phosphatidylcholine (PC) and sarcosine. For each precursor, ten 500 ml Erlenmeyer flasks con- taining 200 m1 of TSB medium and 0.25% (w/v) of the test precursor were prepared. Each flask was inoculated with one of the test cultures (48 hour culture, 1 ml/200 ml medium). The flasks were incubated at the appropriate tempera- ture with shaking on a NBS controlled environment incubator for 48 hours, extracted and prepared for N-nitrosamine analysis using the GC-TEA system. Effect of Incubation Time and Temgerature on NDMA Formation by Growing Bacterial Cultures. Nine 500 ml Erlenmeyer flasks containing 200 ml of TSB media and 0.25% (w/v) dimethylamine-HCl and 0.05% (w/v) nitrite were inocu- lated each with 1 m1 of B. schuylkilliensis. The flasks were incubated in the dark at different temperatures as specified below: Group 1 (3 flasks) at 5°C Group 2 (3 flasks) at 25°C Group 3 (3 flasks) at 32°C 45 One flask from each group was extracted with methylene chloride (as previously described) every 4 days. The extract was concentrated and injected into the GC-TEA system for NDMA analysis. Resting Cells Studies a) Preparation of restinggcells. Cultures were inoculated in 500 ml of the TSB medium in 100 ml Erlenmeyer flasks. The flasks were incubated for 48 hours at the appropriate temperature with shaking. The cells were harvested with centrifugation in a Sorval II refrigerated centrifuge at 6000 rpm for 20 minutes at 2°C. The cell pellet was resuspended in sterile deionized distilled water and recentrifuged. The clean cells were suspended in phosphate buffer (0.2 M, pH 8.0) and stored under refri- geration (4°C) until required. b) Effect of cell concentration on NDMA formation. Four 50 ml suspensions of t. ggtt in phosphate buffer (0.2 M, pH 8.0) were prepared in screw capped milk dilution bottles. The cell concentration was determined as mg protein per m1 and was adjusted to 7, 5.6, 4.2 and 2.8 mg protein/ml of suspension. Nitrite and dimethylamine were added at the levels used in the previous experiment. The bottles were incubated in the dark at 37°C for 5 hours. After incuba- tion, the samples were immediately extracted with methylene chloride and checked for NDMA using GC-TEA. Similar sus- pensions were boiled and treated the same way to serve as 46 blanks. i) Protein determination. Two ml of the bacterial suspension in phosphate buffer (0.2 M, pH 8.0) were digested and analyzed for nitrogen according to standard micro-Kjeldahl procedure described by AOAC (1970). The nitrogen content was multiplied by a factor of 6.25 to determine the protein content. c) Effect of nitrite concentration on NDMA .jgnmgtign. Four 50 m1 g. aureus suspensions (5 mg protein/ ml) in phosphate buffer (0.2 M, pH 8.0) containing 0,25% (w/v) dimethylamine were prepared in screw-capped milk dilution bottles. The nitrite content was adjusted to 0.01, 0.05, 0.1, and 0.2 g per 100 m1 of suspension. The bottles were incubated in the dark at 37°C for 5 hours. After incuba- tion, the cultures were immediately extracted with methylene chloride. The extract was concentrated to 1.0 ml and injected into the GC-TEA system for NDMA identification and quantitation. Extraction of N-Nitrosamines from Bacterial Cultures Direct extraction of liquid bacterial cultures with methylene chloride was used throughout this study. Forty- eight hour cultures were centrifuged inTaSorval II super refrigerated centrifuge for 10 minutes at 2°C and 6000 rpm. The supernatant was collected, the cells were washed two times with sterile, distilled and deionized water and 47 recentrifuged each time. The combined supernatants were saturated with NaCl, extracted with three 50 ml portions of redistilled methylene chloride. The methylene chloride extracts were combined, and the aqueous phase discarded. The combined extracts were dried over anhydrous sodium sulfate and filtered through Whatman No. 42 filter paper. The filtrate was collected in a 1000 ml Kuderna Danish concentration flask, to which was added 2-3 "Boileezers". Snyder columns were fitted to the flasks and the methylene chloride extracts were concentrated to 1 ml by heating the flasks in a steam bath. The concentrated samples were quantitatively recovered into 2 dram screw capped glass vials. The volume was adjusted to 1.0 m1 using nitrogen gas to evaporate excess solvent. The samples were stored at —zo°c until GC-TEA and GC-MS analyses. GC-TEA Analysis Samples, standard NDMA and NPYR were analyzed using the GC-TEA system. The gas chromatograph was a Varian Model 3700 gas chromatograph equipped with a stainless steel column (2 m x 3.0 mm i.d.) packed with 10% Carbowax 20 M + 5% KOH on Chromsorb W (80/100 mesh) obtained from Supelco Inc. (Bellefonte, Pennsylvania). The TEA system was a Thermo Electron thermal energy analyzer model 502 (Thermo Electron Corporation, Analytical Instrument Division, Waltham, MA). 48 G0 Operating Conditions The following conditions were found to be satisfactory and were employed: Carrier gas (nitrogen): 35 ml/min. Injection port temperature: 180°C Column temperature: 180°C, isothermal TEA Conditions Prolyzer furnace temperature: 475°C Trap temperature: -l96°C liquid nitrogen Oxygen flow rate: 10 ml/minute Pressure: 1.5 atmospheres Heated transfer line (between GC and TEA): 190°C Appropriate volumes of standard solutions of NDMA and NPYR and sample extracts were injected into the GC-TEA system. The TEA detector response was recorded on a recorder chart. Peak heights and retention times of stan- dard N-nitrosamines were recorded. The peak heights of all compounds in the samples corresponding to the retention times of the standard N-nitrosamine were measured. The N-nitrosamine contents of the samples were calculated according to the following equation: _ Sph x Stv x Stcon 1000 Stph x Sv x VSC x where S ng N-nitrosamine/total volume of culture 49 Sph = sample peak height (arbitrary units) Stv = standard volume injected (pl) Stcon = standard concentration (ng/ul) Stph = standard peak height (arbitrary units) Sv = sample volume injected (p1) Vsc = total volume of the sample concentrate (ul) Confirmation of the Identity of N-Nitrosamines GC-MS Analysis. The identity of NDMA in some of the positive N-nitrosamine samples from GC-TEA analysis were confirmed by mass spectral analysis according to the follow- ing procedure: The samples were further concentrated to 0.3 ml in a stream of nitrogen. A 3 pl aliquot was injected into the GC-MS system. The mass spectrometer was a Hewlett Packard Model 5985 low resolution quadrupole mass analyzer (Hewlett Packard Corp., Avondale, PA). The gas chromatograph was a Hewlett Packard Model 5840 A equipped with a glass column (2 m x 2 mm i.d.) packed with 10% Carbowax 20 M + 5.0% KOH on Chromosorb W (80/100 mesh). 00 Conditions Carrier gas: helium (35 ml/min) Injection port temperature: 180°C Column temperature: 140-180°C, programmed at 10°C/min with an initial hold of 2 minutes 50 Mass Spectrometer Conditions Ion source temperature: 200°C Electron impact: 70 eV Electron multiplier: Volt: 2000 Start-stop masses: 40-200 UV Irradiation. Irradiation of samples was carried out according to Doerr and Fiddler (1977) with some modifica- tions. Positive N-nitrosamine samples from GC-TEA analysis were exposed to ultraviolet light for 24 hours in a carton paper box laminated with aluminum foil. The light source was a long wave (366 nm) Black-Ray UV lamp Model UVL-21 (Ultra Violet Products Inc., San Gabriel, CA). The extent of photolysis was measured by injecting the samples into the GC-TEA system and analyzing for N-nitrosamines before and after UV irradiation. N-Nitrosation of Sarcosine by Bacteria and Detection of N-Nitrososarcosine by High Performance Liquid Chromatography (HPLC) Five bacterial cultures were used in this study. These were E. coli, §. aureus, t. bulgaricug, M. thermosphactum and Pg. fluorescens. A 1 ml culture was inoculated into 200 ml of TSB medium containing 0.25% (w/v) sarcosine and 0.05% (w/v) NaNOz. The cultures were incubated at the appropriate temperatures for 48 hours with shaking and aeration (except for t. bulgaricus). The cultures were 51 filtered through a Millipore filter (0.45 u) and the supernatants collected and refiltered. The pH of the supernatants was adjusted to 3.1 using H3P04. A 50 ul aliquot of the supernatants was injected into the HPLC system for identification and quantitation of N-nitrosar- cosine. The chromatographic system consisted of a Waters Asso- ciates liquid chromatograph, equipped with a chromatography pump Model 6000 A, and a uBondapak C18 reverse phase column (32 cm x 6 mm i.d.), a Model 440 absorbance detector and strip chart recorder (Linear Instruments). Appropriate volumes of a Standard N-nitrososarcosine solution (2 mg/100 ml) and samples were injected into the HPLC and the samples eluted with a 0.1 M sodium phosphate buffer (pH 3.0) at a flow rate of 1.0 ml/min, and detected by ultraviolet absorption at 254 nm. The UV detector response was recorded. Peak heights and retention times of a standard N-nitrososarcosine solution were recorded. The peak heights of compounds in the samples corresponding to the retention time of standard N-nitrososarcosine were measured. The N-nitrososarcosine content of samples were calcu- lated from a standard curve of peak height vs standard N-nitrososarcosine concentration. 52 Cid-deed Processing A one half lamb carcass weighing approximately 18 lbs was obtained from a commercial meat distributor in the East Lansing area 10 hours after slaughter. The carcass was cut into three sections, front leg, middle section and hind leg, and deboned. Each section was divided longi- tudinally into four equal pieces. Four representative samples (A, B, C and D) were pooled from the three sections (1350 g each). The meat in each sample was further sliced to about 2 cm in thickness. The first sample (A) was heavily salted by rubbing an unknown amount of dry salt (refined salt flakes) on the meat slices until a satisfactory product was obtained. The amount of salt used (126 g) was calculated backwards by weighing the remainder of a pre- weighed stock. Samples 8, C and D were treated as follows: Treatment Salt 8 126 g refined salt + 120 mg/kg of nitrite C 126 g refined + 500 mg/kg of nitrate D 126 g crude salt The samples were hung on a rope in the Open air in the sun for 3 days. Representative samples from each treatment were fried in olive oil to a medium "doneness" on a slow flame. The temperature of the frying oil was allowed to increase from room temperature to 190°C in approximately 7 minutes, after which the frying was discontinued. The fried gid-deed was removed from the oil and placed on a 53 filter paper to remove excess oil. Both the salted-sundried and the fried samples were analyzed for the presence of N-nitrosamines using vacuum distillation and GC-TEA as described later. Free Amino Acid Analysis of the Meat Samples samples from fresh and salted-sundried lamb meat were analyzed for their free sarcosine and proline contents according to the procedure of Clark gt _t. (1966), as modified by Gray and Collins (1977). The meat samples were homogenized in a Waring blender with 1000 ml of 3% 5-su1- fosalicylic acid and were immediately centrifuged for 10 min in Sorval II super refrigerated centrifuge at 4000 rpm. The supernatant was removed and the residue was again extracted with 500 m1 of the sulfosalicylic acid solution and centrifuged as before. The combined supernatants were freeze-dried and the residue dissolved in 100 ml of citric acid buffer (0.2 M) at a pH of 2.1. The sample solution was extracted with 100 m1 of n-hexane to remove residual lipids, filtered through a Millipore filter (0.2 p) and stored at -20°C until analysis. A Dionex amino acid analyzer fitted with a DC-4 resin (Durrum) cation exchange column (column bed 26 cm x 3 mm) was used for amino acid determination. Amino acids in the samples and reference amino acids (including proline) were eluted using a lithium citrate buffer system and reacted with ninhydrin to produce the 54 colored chromogen for quantitation. Vacuum Distillation and Extraction of N-Nitrosamines in the Gid-Deed Samples Twenty-five grams of the ground meat samples (fried and unfried) were distilled under reduced pressure according to the procedure described by Robach gt _t. (1980) using paraffin oil as the distillation medium and ammonium sul- famate (0.5 g/25 g sample) to prevent N-nitrosamine formation during the distillation step. Sample distillates were recovered in vacuum traps immersed in liquid nitrogen. The distillation continued until an internal temperature of 115°C was reached in the distillation flasks. The distil- lates were allowed to thaw in the dark and were extracted using methylene chloride as described by Robach gt gt. (1980). TM (Thermo Electron Corp., Waltham, Mass.) were Preptubes used to remove H20 and reduce impurities in the methylene chloride extract. The extracts were concentrated as before and injected into the GC-TEA system for N-nitrosamine analysis. Nitrite Analysis Nitrite analyses were carried out according to the stan- dardTAssociation of Official Analytical Chemists (AOAC) proce- dure (T970), with the following modification: N-T-naphthyl- ethylene-diamine dihydrochloride was used to produce the 55 colored chromogen instead of a-naphthylamine since the latter is a recognized carcinogen. RESULTS AND DISCUSSION Separation and Identification of N-Nitrosamines bngC-TEA The chromatographic separation of volatile N-nitrosa- mines by the GC-TEA system using TEA as a detector provides a very powerful tool for the separation and identification of these compounds. The method is relatively specific, and requires a minimum of purification steps (Fine gt gt., 1975a). The TEA detector is extremely sensitive to N- nitrosamines, and is almost non-responsive to other impuri- ties that might be present in the sample (Fine gt gt., 1975b). A standard mixture of NDMA and NPYR in methylene chloride was injected into the GC-TEA system whenever sample extracts were analyzed. Usually 2.5 ul of the standard mixture containing 10 ng of each of NDMA and NPYR (4 ng of each/1 01) were used. Figures 1 and 2 illustrate typical chromatograms obtained when standard N-nitrosamines and sample extracts (in methylene chloride) were injected, under the employed conditions, into the GC-TEA system. 56 57 NDMA TEA RESPONSE :=-— NPYR r/ L, _._.J L... 012 3 4 S 6 7 8 TIME(min) Figure l. GC-TEA chromatogram of standard N-nitroso- dimethylamine and N-nitrosopyrrolidine. 58 NDMA TEA RESPONSE L_l 1 J l .1 01234 TIME ( min) Figure 2. GC-TEA chromatogram of a typical methylene chloride extract of a bacterial culture containing precursors of N-nitrosodimethylamine. 59 Confirmation of the Identity of N-Nitrosamines by GC-MS Analysis and UV Irradiation The identity of NDMA in some of the positive N-nitro- samine samples was confirmed by mass spectral analysis and UV irradiation. The fragmentation patterns of NDMA in the samples were similar to those of authentic NDMA and to the fragmentation patterns of the N-nitrosamine reported in literature (Kawabata gt gt., 1977). Typical spectra of NDMA are shown in Figure 3. Photolysis of the NDMA and N-nitrosodibutylamine (NDBA) in the samples was evident when the methylene chloride extracts were subjected to UV irradiation. Doerr and Fiddler (1977) have stated that, for low levels of N- nitrosamines that can not be confirmed by mass spectral analysis, photolysis at 366 nm of a small portion of the sample concentrate offers a simple, rapid and sensitive method for the presumptive evidence of N-nitrosamines. Therefore, the formation of N-nitrosamines in the bacterial cultures can be more or less positively identified by the combination of TEA analysis and UV photolysis. NDMA Formation by Growigngacterial Cultures from Dimethyla- mine and Nitrite The bacterial cultures were grown in 200 ml of TSB medium containing 0.25% (w/v) dimethylamine and 0.05% (w/v) sodium nitrite. The samples were extracted after 48 hours 60 Figure 3. Mass spectra of: (A) N-nitrosodimethylamine standard, and (B) N-nitrosodimethylamine isolated from bacterial cultures containing dimethylamine and nitrite/or nitrate. 61 02. cc ow E 2V NV '8% .2.“ -85 M ..omw .. o2 E mu Nv 8' ‘3 8| )IVEld 35178 :10 % l g I l g a 62 of incubation and the amount of NDMA produced in the incuba- tion medium was determined by the GC-TEA system (Table 6). The Table 6. NDMA formation by growing bacterial cultures in 200 ml of tryptic soy broth containing dimethyla- mine (0.5 g) and nitrite (0.1 g). Organism ug/ZOONTITJTTACulturea (032°XfN3Ug? E. coli E-2 384.0 0.056 E. coli K-12 342.9 0.055 t. bulgaricus 1481.5 0.236 E. thermosphactum 173.2 0.028 3. fluorescens 11.8 0.002 E. schuylkilliensis 42.1 0.007 g. schuytkilliensis 23.1‘ 0.004 3. ggttgg biotype A 12.9 0.002 3. ggttgg 14.2 0.002 t. g_reus 297.1 0.004 Control (pH 7.2) 6.1 0.001 aCorrected for control determination highest yield of NDMA was produced by t. tglgaricus being 1481.4 pg, followed by the two t. ggtt species and g. aureus which produced 384.0, 342.9 mmd297.1 pg, respectively. The lowest N-nitrosamine yield was obtained by 3. putida biotype A, (12.92 pg). These results correlate very well with published literature regarding N-nitrosamine formation by bacteria. Hawksworth and Hill (1970) showed that out of 63 ten strains of S. ggtt tested, five were able to form N- nitrosamines when incubated aerobically in a nutrient broth medium containing nitrate or nitrite and the secondary amines, dimethylamine, diethylamine, diphenylamine, piperi- dine, pyrrolidine and N-methylaniline. Similarly, NDMA has been shown to be produced from dimethylamine and nitrite by S. aureus, S. ggidirmidis and Psuedomonas species (Mills and Alexander, 1976; Ishiwata _t _t., 1976; Uibu and Bogovski, 1978). The starting pH of the culture medium was 7.2 i 0.1 and the final pH varied with bacterial species tested. For E. tgtt species, the final pH was 5.6. Similarly, the final pH of t. bulgaricus and M. thermosphactum was 5.4 and 5.7, respectively. All other species had final pH values above 7.2. These pH values indicate that chemical N-nitrosation catalyzed by acid production in the culture media may have been partly responsible for N-nitrosamine formation. Mirvish (1970) showed that for dimethylamine and nitrite, under standard conditions of temperature and total species concentration, a maximum NDMA formation was obtained at pH 3.4. The percent yield decreased on both sides of this optimum pH. He also reported that over a pH range of 9 to 5, the rate of N-nitrosation increased 10 fold for every unit drop in pH. Accordingly, t. bulgaricus and S. coli are expected to produce more NDMA in the incubation medium than the other species used. This was borne out by the 64 experimental data obtained. NDMA Formation gy Resting Cells at pH 8.0 To investigate the catalytic nature of N-nitrosamine formation from dimethylamine and nitrite by bacterial species and to ascertain whether the bacterial catalysis of N-nitrosamine formation is merely the result of a change in pH or some other phenomenon, resting cells of the test organisms are suspended in 100 ml of 0.2 M phosphate buffer at pH 8.0. Dimethylamine and nitrite were added at the same level as in the growing cultures (0.05% (w/v) nitrite and 0.25% (w/v) dimethylamine). The reaction mixtures were incubated for 5 hours at the optimum temperatures for the bacterial suspensions. The total NDMA formed in each suspension was determined, after correcting for the blank determinations (Table 7). These data indicate that NDMA formation from dimethylamine and nitrite was catalyzed by the presence of bacteria, even at pH 8.0. This is a pH value, at which chemical N-nitrosamine formation is minimal (Mirvish, 1970). Therefore, it could be assumed that another catalytic mechanism is involved in N-nitrosamine formation by these bacterial species. Klubes (1972), Ayanaba and Alexander (1973), and Kunisaki and Hayashi (1979) reported that boiled bacterial cells including those of S. ggtt B were unable to N-nitrosate dimethylamine in the presence of nitrite. However, NDMA was produced in the 65 Table 7. NDMA formation by resting bacterial cells in 100 ml phosphate buffer (0.2 M, pH 8.0) con- taining 0.25 g dimethylamine and 0.05 g sodium nitrite. Organism pg/lOONETABuffer % Conversiona (DMA—eNDMA) S. coli E-2 0.089 2.8 S. coli K-12 0.035 1.2 t. bulgaricus 0.120 3.8 fl. thermosphactum 0.510 16.2 S. fluorescens 0.533 17.0 S. schuytkilliensis 0.055 1.8 S. schuylkilliensis 0.083 2.6 S. putida biotype A 0.143 4.6 _P_. gum 0.238 ' 7.5 S. _greus 0.125 4,0 Control (no culture) 0-007 0.24 a x 105 presence of living cells. They concluded that N-nitrosamine catalysis by bacteria was enzymatic in nature. 0n the other hand, Collins-Thompson _t _t. (1972) and Yang _t _t. (1977) showed that boiled cells were equally active in catalyzing N-nitrosamine formation from secondary amines and nitrites and concluded that the N-nitrosation reaction is non-enzy- matic. Data presented in Tables 6 and 7 also indicate that 66 under growing conditions, t. bulgaricus, S. ggtt species and S, ggtggs,respectively,produced the greatest amount of NDMA after incubating for 48 hours in a TSB medium. At pH 8.0 in a 0.2 M phosphate buffer solution and after 5 hours of incubation, a major shift in N-nitrosamine formation was observed. Under these conditions, S. fluorescens and fl. thermosphactum were shown to be the most active in catalyzing NDMA formation from dimethylamine and nitrite. This pheno- menon indicated that a pH-dependent mechanism is primarily involved in the N-nitrosation reaction when growing bacterial cultures are involved. Effect of Nitrite Concentration on NDMA Formation Resting cells of S. aureus species were incubated for 5 hours in 50 ml.of 0.2 M phosphate buffer (pH 8.0) contain- ing a fixed concentration of dimethylamine (0.25% w/v) and increasing nitrite concentrations (0.01, 0.05, 0.1 and 0.2% w/v). N-Nitrosamine analysis of the incubated suspensions indicated that NDMA formation increased with increasing nitrite levels (Table 8). Furthermore, NDMA formation was doubled as the nitrite concentration was increased from 0.01% to 0.05% and increased four times when the nitrite concentration was increased from 0.05% to 0.1%. However, NDMA formation was increased only by a factor of 0.5 as the nitrite concentration was increased from 0.1% to 0.2%. The N-nitrosation reaction is second order with respect to 67 Table 8. Effect of nitrite concentration on NDMA formation by S, aureus using a fixed dimethylamine concen- tration at 37°C and pH 8.0 for 5 hours. Concentration NDMA of nitrite (%) ng/50 ml buffer % Conversiona (DMA-aNDMA) 0.01 103.23 6.6 0.05 225.22 14.6 0.1 1013.51 64.9 0.2 14358.11 91.9 ax lO5 nitrite concentration, i.e., the rate of the reaction is increased 4 times with doubling of the nitrite concentration (Mirvish, 1970). In this study, this criterion was only evi- dent when the concentration of nitrite was increased from 0.05% to 0.1%. This is probably due to the fact that N-nitrosation of secondary amines in a bacterial system operates under kinetic rules different from those followed in a chemical model system. Kunisaki and Hayashi (1979) reported that the formation of N-nitrosamines by the resting cells of S. ggtt B was proportional to the incubation time and cell concentration. They also indicated that the reaction followed Michaelis-Menten kinetics and was inhibi- ted by high concentration of the substrates, dimethylamine and nitrite. 68 Effect of Cell Concentration on NDMA Formation Resting cells of S. ggtt K-12 were suspended in a 50 m1 of phosphate buffer (0.2 M, pH 8.0) containing 0.25% (w/v) dimethylamine and 0.05% (w/v) nitrite. The cell concentration was adjusted to 7.0, 5.6, 4.2, and 2.8 mg protein/ml. Data in Table 9 show the total amount of NDMA Table 9. Effect of cell concentration of S. coli K-12 on NDMA formation at constant pH (8.0) using fixed concentrations of nitrite and dimethyla- mine at 37°C for 5 hours. 11 ' . °°(mg°g:§:2§;?$;?n ng75UAml % ConverSTod‘(DMA-+NDMA) 7.0 899 5.75 5.6 577.8 3.70 4.2 486.1 3.11 2.8 355.8 2.28 Blank (Boiled 7.0 166.7 1.07 cells) ax 104 accumulated in each suspension. These results indicated that NDMA formation increased with increasing cell concen- tration. NDMA was also detected in the boiled cell suspen- sion (blank or control), the amount being approximately 18.5% of that produced by the living cells. These results indicated that a major proportion of NDMA formed by S. coli K-12 was catalyzed by a heat sensitive mechanism, possibly 69 enzymes. NDMA Formation by Growing Bacterial Cultures from Dimethylamine and Nitrate Data presented in Table 10 indicate that out of the 10 bacterial species tested, only 3 were capable of producing Table 10. NDMA formation by growing bacterial cultures from sodium nitrate (0.5 g) and dimethylamine (0.5 g) in 200 ml TSB. NDMA Organism “9,200 ml culture % Conversiona (DMA—eNDMA) S. coli E-2 26.57 4.24 S. coli K-12 32.85 5.25 t. bulgaricus -- , -- fl. thermosggactum traces -- S. fluorescens 0.21 0.03 S. schgylkilliensis 0.14 0.02 S. schuyjkilliensis 0.12 0.02 S. ggttgg biotype A 0.22 0.04 S. putida 0.05 0.01 S _greus 320.0 51.11 ax 103 appreciable amounts of NDMA when the growing cultures were incubated in TSB media in the presence of nitrate and dimethylamine. These species were: S. coli E-2, S. coli K-12 and S. aureus. Other cultures produced relatively 70 smaller amounts similar to those obtained in the control (blank). The production of NDMA by these bacterial species from dimethylamine and nitrate indicated that these organisms are capable of reducing nitrate to nitrite which will sub- sequently N-nitrosate dimethylamine. Similar results have been reported by Hawksworth and Hill (1970) and Ishiwata gt _t. (1976). Hawksworth and Hill (1970) showed that out of ten strains of S. ggtt tested, five were able to form N-nitrosamine when incubated aerobically in a nutrient broth medium containing nitrate and secondary amines. S. aureus has also been shown to produce NDMA when incubated in brain heart infusion (BHI) containing dimethylamine and nitrate (Ishiwata gt gt., l976). Nitrate reduction by bacteria through nitrate reductase activity results in the accumula- tion of nitrites; however, certain bacterial species have been shown to reduce nitrate to gaseous nitrogen (Atlas and Bartha, 1981). In this case nitrite does not accumulate, and NDMA is not expected to be formed in appreciable quantities, N03-——+ N02 NO'-——+ N 3 2 even if dimethylamine is present at a high concentration in the incubation medium. Similarly, NDMA will not be formed if the organisms are not endowed with nitrate reductase activity. Previous studies have indicated that the actual 71 N-nitrosating species can be one of the following depending on N-nitrosation conditions: nitrous anhydride (N203), nitrous acidium ion (NO+), nitrosyl halide (NOX) or nitrosyl thiocyanate (NOCNS) (Challis and Butler, 1968; Mirvish, 1972; Mirvish, 1975). Effect of Incubation Time and Temperature on NDMA Formation ty P. schuylkilliensis Growing Cultures Three sets of growing cultures of S. schuylkilliensis were prepared. Each set contained three 500 ml Erlenmeyer flasks. Each flask contained 200 ml of TSB medium to which were added 0.25% (w/v) dimethylamine and 0.05% (w/v) sodium nitrite along with 1.0 m1 of a S. gghuylkilliensis active culture. The first set was incubated at 5°C. The second and the third sets were incubated at 25°C and 32°C, respec- tively. A flask from each set was removed every four days, and the contents extracted and analyzed for NDMA. Data in Table 11 show the total NDMA formed in each flask. These data indicated that N-nitrosamine formation by S. schuylkil- ttggStS was, as expected, dramatically increased by increas- ing the incubation temperature. The NDMA formed after 4 days of incubation increased approximately Six times as the incubation temperature was increased from 5 to 25°C, and 67 times as the incubation temperature was increased to 32°C. However, these differences were less pronounced after 8 to 12 days of incubation. These results also indicated 72 Table 11. Effect of incubation time and temperature on NDMA formation by S, schgylkilliensis growing in 200 ml of TSB medium containing 0.T'g nitrite and 0,5 g dimethylamine. Days of incubation pg NDMA formed/200 ml culturea 5°c 25°C 32°C 4 0.5 3.0 33.6 8 0.32 0.63 7.51 12 0.32 0.74 1.77 aValues are the average of two replicates. that NDMA was reduced in the 25 and 32°C cultures as the incubation time was extended beyond 4 days. However, the quantity of NDMA in the 5°C cultures remained almost unchanged as the incubation time was extended to 8 and 12 days. As for other chemical reactions, N-nitrosation reac- tion rates have been shown to be doubled for every 10°C rise in temperature (Foreman and Goodhead, 1975). The results of this study indicated a similar trend. Prolonged storage conditions have been reported to increase N-nitrosamine formation even at low temperature (Ender gt gt., 1967). In the present study, longer incuba- tion (8 and 12 days) resulted in lower levels of NDMA, especially at the 25°C and 32°C incubation temperatures. This loss of NDMA could be due to degradation of the pre- formed N-nitrosamine by S. schuylkilliensis or by volatili- zation of the NDMA to the atmosphere. In both cases, the 73 loss is expected to be much higher at 25°C and 32°C incuba- tion temperatures. Degradation of preformed NDMA by various bacteria have been reported by Klubes gt gt. (1972) and Rowland and Grasso (1975). Both reported a loss of approximately 5% of the N-nitrosamine when incubated with bacteria under standard conditions and pH 7.0 for 20 hours. Similar results were reported earlier by Hawksworth and Hill (1971) who demon- strated that NDMA and NDEA were degraded to the parent secondary amine and nitrite by washed-cell suspensions of five S. ggtt strains (out of ten strains tested), 3/10 of the clostridia and 3/10 of non-spore forming anaerobes tested. They also indicated that the enzyme responsible for the degradation was of low activity (maximum level of breakdown observed after overnight incubation was only 0.025%), was located in the cytoplasmic material of the cell and had a pH optimum range of 7 to 8. NDMA Formation ty Growing Bacterial Cultures from Trimethyla- mine and Trimethylamine-N-Oxigg Bacterial cultures were incubated in TSB media con- taining 0.25% (w/v) trimethylamine or trimethylamine oxide and 0.05% (w/v) sodium nitrite. Results of NDMA analysis indicated that most of the bacterial cultures had a higher N-nitrosamine content when incubated with trimethylamine- oxide than with trimethylamine after 48 hours of incubation 74 (Table 12). The highest conversion was observed in the S. aureus culture containing trimethylamine oxide. Both trimethylamine and trimethylamine oxide have been implicated in NDMA formation when heated in chemical model systems in the presence of nitrite (Scanlan gt _t., 1974; Ohshima and Kawabata, 1978). The latter group reported that NDMA was produced in yields as high as 53% from trimethylamine and 55% from trimethylamine oxide when the amines were reacted with nitrites at pH 3.0 and 100°c for 2 hours. However NDMA yields were much lower (2-3% from trimethylamine and 0.02% from trimethylamine oxide) at pH 6-7 under the same heating conditions. Similarly, trimethylamine has also been shown to be converted to dimethylamine which is subsequently N-nitro- sated to NDMA in the presence of bacteria and nitrates or nitrites (Ayanaba gt gt., 1973; Ayanaba and Alexander, 1973; Maduagwu and Bassir, 1979). Similar to nitrate, trimethylamine oxide has been shown to serve as a terminal electron acceptor in anaerobic respi- ration of bacteria in which a variety of hydrogen donors are oxidized at the expense of trimethylamine oxide which is reduced to trimethylamine (Strom, 1979). Therefore, trimethylamine oxide is ultimately reduced to trimethylamine, then possibly demethylated to dimethylamine, and N-nitrosated to NDMA via bacterial activity. This process takes place as bacteria attain higher counts in the incubation media. 75 Table 12. NDMA formation by growing bacterial cultures from triamines (0.5 g) and nitrite (0.1 g) in 200 m1 of tryptic soy broth. pg NDMA/200 ml culture % Conversiona % Conversiona TMA (TMA-*NDMA) TMA-0 (TMA-'9 NDMA) S coli E-2 Traces -- 0.14 0.31 S coli K-12 Traces -- 0.28 0.63 t. bulgaricus 1.39 2.6 2.43 5.43 S. thermosghactum 0.33 0.62 0.53 1.18 S. fluorescens Trace -- 0.20 0.45 S. schgylkilliensis 0.26 0.49 0.12 0.27 S. schuytkilliensis 0.14 0.26 0.20 0.45 S. putida biotype A Traces -- 0.17 0.38 S. putida 0.13 "0.24 0.11 0.25 S. aureus 0.13 0.24 107.39 240.05 TMA = Trimethylamine-hydrochloride TMA-0 = Trimethylamine-N-oxide: dihydrate ax 104 76 Compared to trimethylamine oxide, trimethylamine is extremely volatile (bp. 3.7°c). This may explain the higher yields of NDMA from trimethylamine oxide as opposed to those obtained from trimethylamine. Mechanism of NDMA Formation from TertiarytAmines Two possible mechanisms have been suggested for the formation of NDMA from tertiary amines and nitrite (Loeppky and Smith, 1967; Lijinsky gt_ 1., 1972; Sander gt 1., 1975). The first involves the oxidative cleavage of one N-R bond before N-nitrosamine formation. The second mecha- nism involves nonoxidative cleavage of one N-R bond when the R group is in an oxidation state appropriate for its removal by hydrolysis (Lijinsky _t gt., 1972). In both mechanisms, the tertiary amines are converted to secondary amines before the final N-nitrosation step. This conversion is suggested to take place as follows: the tertiary amine is directly attacked by the nitrous acidium ion to form an adduct which decomposes spontaneously to form an iminium ion. In the presence of water, the iminium ion is hydrolyzed to the secondary amine and an aldehyde. The secondary amine is N-nitrosated by a nitrite ion to form a N-nitrosamine. An alternative mechanism was proposed later by Ohshima and Kawabata (1978) in which NDMA may be formed directly from trimethylamine or trimethylamine oxide by a pathway not involving secondary amines. In this mechanism, the iminium 77 ion may undergo nucleophilic attack by the nitrite ions to form an unstable complex which directly decomposes to NDMA and formaldehyde. NDMA Formation by Growing Bacterial Cultures from Choline and Choline-Containing Compounds Data presented in Table 13 indicate that NDMA, in the range of trace amounts to 1.23 pg,_were produced when the bacterial cultures were incubated for 48 hours in 200 ml of TSB medium containing 0.05% (w/v) nitrite and 0.25% (w/v) of each of these compounds, separately. Again, the total NDMA accumulated varied with the bacterial species and the compound used. However, higher N-nitrosamine yields were obtained when lecithin was utilized as a precursor. Choline, phosphorylcholine and lecithin have been cited as possible precursors for NDMA in foods, and were shown to result in NDMA formation when heated with nitrite in a chemical model system (Gray gt gt., 1978). On the other hand, N-nitrosamine formation from these compounds via microbial activity has not been reported so far. However microbial degradation of choline and choline compounds have been reported to result in dimethylamine formation (Asatoor and Simenhoff, 1965; Hawksworth, 1970; Hawksworth and Hill, 1971). As far as NDMA formation is concerned, lecithin, when used as a precursor, gave higher NDMA yields with most bacteria than did choline or phosphorylcholine. 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