N-nitrosoproline Research Articles

Photomutagenicity of N-nitrosoproline dissolved in non-aqueous solvent, oleic acid

In the present study, we investigated the genotoxicity of the active products formed from N-nitrosoproline (NPRO) dissolved in oleic acid following ultraviolet A (UVA) irradiation, bypassing the need for metabolic activation. We previously demonstrated the photomutagenicity of NPRO dissolved in a phosphate-buffered solution. It has been suggested that the association of the nitrosamine group with acid ions facilitates rapid photodissociation and photoactivation. We hypothesized that NPRO’s inherent carboxyl group may mimic an acid, inducing photodissociation and photomutagenicity, even in a non-aqueous solvent lacking acidic ions. Following UVA irradiation, NPRO dissolved in oleic acid exhibited a dose-dependent mutagenic activity. Similar results were obtained when NPRO was dissolved in linoleic acid and triolein. Nitric oxide formation, which is dependent on NPRO concentration, is accompanied by mutagenic activity. The mutagenicity spectrum obtained in response to NPRO irradiation followed the absorption curve of NPRO dissolved in oleic acid. Irradiated NPRO in oleic acid displayed relative stability, retaining approximately 18, 36, and 63 % of initial mutagenicity after 10 days of storage at 25, 4, and −20 °C, respectively. Thus NPRO stored in a fatty environment undergoes photoactivation upon irradiation, leading to genotoxicity.

Isolation of new photoadducts from UVA-irradiated N-nitrosoproline with 2'-deoxyadenosine and characterization of photoadducts from DNA irradiated with N-nitrosoproline

N-nitrosoproline (NPRO) is formed from nitrosation of proline and has been reported to be non-carcinogenic and non-mutagenic. However, earlier studies in our laboratory showed that pre-irradiated NPRO can be converted to a mutagenic form. We previously investigated the reaction of NPRO with dA or dG under UVA irradiation and identified the formation of 2-pyrrolidyl-dA adducts (P1 & P2) and 8-pyrrolidyl-dG adducts (G1 & G2) as well as four known modified nucleosides, although several peaks found in the HPLC profiles of UVA-irradiated mixtures of dA and NPRO remain unidentified. In the present study we isolated new photoproducts from irradiated mixtures of dA and NPRO and identified (R)- and (S)-8-(2-pyrrolidyl)-2′-deoxyadenosine (A1 and A2) as products by MS and NMR. We also investigated the photoadducts formed in DNA treated with NPRO under UVA irradiation, and detected A1 and/or A2 (probably both), P1, P2, G1 and/or G2, and 8-oxodG as products. Under anaerobic conditions, formation of A1 and A2 was greater than that under aerobic conditions, suggesting that photo-reactions comprising pyrrolidyl radical with dA may increase under anaerobic conditions given reduced competition with oxidative photo-reactions which may decompose pyrrolidyl-dA adducts.

Isolation and identification of photoproducts from UVA-irradiated mixture of N-nitrosoproline with 2'-deoxyadenosine

N-Nitrosoproline (NPRO) is known to form endogenously from sodium nitrite and the amino acid proline, and is thought to be non-mutagenic and non-carcinogenic. However, earlier studies in our laboratory showed that irradiated NPRO can be converted directly to a mutagenic compound upon UVA irradiation. We previously investigated the mutagenic spectrum of NPRO on M13mp2 DNA with UVA irradiation, and found a hot spot comprising AT to GC, where there might be a particular photoreaction of adenine residues with NPRO. In the present study, we investigated the reaction of NPRO with 2'-deoxyadenosine (dA) under UVA irradiation, and identified the chemical structures of the photoproducts. Photoproducts from NPRO and dA were isolated and analyzed with HPLC, MS and NMR. (R)-, (S)-2-(2-pyrrolidyl)-2’-deoxyadenosine (P1, P2) and deoxyinosine (dI) were identified from the mixture of dA and NPRO irradiated with UVA. Results suggest that UVA-activated NPRO produces pyrrolidyl cations which react with dA to generate pyrrolidyl-dA adducts. Production of P1 and P2 from irradiated NPRO with dA increased under N2 saturation compared to those under air saturation, while production of P1 and P2 from irradiated solution under O2 saturation was lower compared to that under air. These results suggest that a Type-I mechanism is involved in the photoproduction of P1 and P2. Furthermore, reactive oxygen species generated during photo-reaction in the presence of O2 may act to destroy the activated form of irradiated NPRO to reduce the formation of P1 and P2. The formation of dI in the irradiated mixture of NPRO with dA might mediate NO release from irradiated NPRO.

Mutation spectrum resulting in M13mp2 phage DNA exposed to N-nitrosoproline with UVA irradiation

N-nitrosoproline (NPRO) is endogenously formed from proline and nitrite. In an effort to delineate the mechanism of NPRO-induced photomutagenicity, we investigated the mutagenic spectrum of NPRO on M13mp2 DNA with UVA irradiation. Following exposure to NPRO and UVA, the mutation frequency increased significantly in an NPRO and UVA dose-dependent manner. The sequence data derived from seventy of the mutants indicated that mutagenesis resulted mainly from an increase in single-base substitutions, the most frequent being GC to CG transversions. Non-clustering of the GC to CG mutations suggests that NPRO+UVA damage to DNA is random. These transversions may be caused by guanine adducts in DNA or in part by oxidatively modified guanine in DNA exposed to NPRO and UVA.

Urinary levels of N-nitroso compounds in relation to risk of gastric cancer: findings from the shanghai cohort study.

Background N-Nitroso compounds are thought to play a significant role in the development of gastric cancer. Epidemiological data, however, are sparse in examining the associations between biomarkers of exposure to N-nitroso compounds and the risk of gastric cancer.MethodsA nested case-control study within a prospective cohort of 18,244 middle-aged and older men in Shanghai, China, was conducted to examine the association between urinary level of N-nitroso compounds and risk of gastric cancer. Information on demographics, usual dietary intake, and use of alcohol and tobacco was collected through in-person interviews at enrollment. Urinary levels of nitrate, nitrite, N-nitroso-2-methylthiazolidine-4-carboxylic acid (NMTCA), N-nitrosoproline (NPRO), N-nitrososarcosine (NSAR), N-nitrosothiazolidine-4-carboxylic acid (NTCA), as well as serum H. pylori antibodies were quantified in 191 gastric cancer cases and 569 individually matched controls. Logistic regression method was used to assess the association between urinary levels of N-nitroso compounds and risk of gastric cancer.ResultsCompared with controls, gastric cancer patients had overall comparable levels of urinary nitrate, nitrite, and N-nitroso compounds. Among individuals seronegative for antibodies to H. pylori, elevated levels of urinary nitrate were associated with increased risk of gastric cancer. The multivariate-adjusted odds ratios for the second and third tertiles of nitrate were 3.27 (95% confidence interval = 0.76–14.04) and 4.82 (95% confidence interval = 1.05–22.17), respectively, compared with the lowest tertile (P for trend = 0.042). There was no statistically significant association between urinary levels of nitrite or N-nitroso compounds and risk of gastric cancer. Urinary NMTCA level was significantly associated with consumption of alcohol and preserved meat and fish food items.ConclusionThe present study demonstrates that exposure to nitrate, a precursor of N-nitroso compounds, may increase the risk of gastric cancer among individuals without a history of H. pylori infection.

Formation and mitigation of N-nitrosamines in nitrite preserved cooked sausages

Literature on formation and mitigation of N-nitrosamine (NA) and especially non-volatile NA (NVNA) in meat products is scarce and the present study is therefore a relevant contribution to the field. We found positive correlation between the levels of N-nitrosopiperidine (NPIP), N-nitrosohydroxyproline (NHPRO), N-nitrosoproline (NPRO), N-nitrosothiazolidine-4-carboxylic acid (NTCA) and N-nitroso-2-methyl-thiazolidine-4-carboxylic acid (NMTCA) and the amount of nitrite added to cooked pork sausages. The levels studied were 0, 60, 100, 150, 250 and 350mgkg−1. The levels of N-nitrosodimethylamine (NDMA) and N-nitrosopyrrolidine (NPYR) remained at or below limit of quantification. Erythorbic acid inhibited the formation of NHPRO, NPRO, NPIP and NTCA. This inhibition was for NTCA and NMTCA counteracted by addition of free iron. Ascorbyl palmitate had less inhibitory effect than erythorbic acid and a combination of the two provided no further protection. Increasing the black pepper content increased the levels of NPIP and NMTCA. Only slight effects of increased fat content and addition of tripolyphosphate were observed.

Occurrence of volatile and non-volatile N-nitrosamines in processed meat products and the role of heat treatment

Most of the available data on the occurrence of N-nitrosamines (NA) in processed meat products have been generated in the 1980s and 1990s and especially data on the occurrence of non-volatile NA (NVNA) are scarce. Therefore we have studied the levels of volatile nitrosamines (VNA) and NVNA in processed meat products on the Danish market (N = 70) and for comparison also products on the Belgian market (N = 20). The effect of heat treatment on the NA levels, in selected samples, was also studied, in order to enable an evaluation of how preparation before consumption affects the levels of NA. For the Danish products the mean levels of the VNA were generally low (≤0.8 μg kg−1), whereas the mean levels of the NVNA were considerably higher (≤118 μg kg−1). Slightly higher mean levels were indicated for the Belgian products (i.e. VNA ≤1.5 μg kg−1 and NVNA ≤270 μg kg−1). The sums of VNA were higher than 10 μg kg−1 for one Danish sample and two Belgian samples. Levels of up to 2000 and 4000 μg kg−1 of N-nitroso-thiazolidine-4-carboxylic acid (NTCA) an NVNA occurred in the Danish and the Belgian samples, respectively. The majority of the Danish processed meat products contain NVNA but also VNA occur. The levels of NA are comparable with those reported in previous and recent studies; however the frequency in which they are found may be lower and thereby also the mean contents. The levels of N-nitrosopiperidine (NPIP) increased by frying and baking, whereas varying impacts were observed for N-nitrosoproline (NPRO), N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR), N-nitrosodiethylamine (NDEA) and N-nitrosomethylaniline (NMA) depending on the type of product and/or the heat treatment. The levels of the NVNA, NTCA and N-nitroso-2-methyl-thiazolidine 4-carboxylic acid (NMTCA) decreased after frying and baking.

Simultaneous determination of volatile and non-volatile nitrosamines in processed meat products by liquid chromatography tandem mass spectrometry using atmospheric pressure chemical ionisation and electrospray ionisation

A sensitive, selective and generic method has been developed for the simultaneous determination of the contents (μgkg−1 range) of both volatile nitrosamines (VNA) and non-volatile nitrosamines (NVNA) in processed meat products. The extraction procedure only requires basic laboratory equipment and a small volume of organic solvent. Separation and quantification were performed by the developed LC–(APCI/ESI)MS/MS method. The method was validated using spiked samples of three different processed meat products. Satisfactory recoveries (50–130%) and precisions (2–23%) were obtained for eight VNA and six NVNAs with LODs generally between 0.2 and 1μgkg−1, though for a few analyte/matrix combinations higher LODs were obtained (3 to 18μgkg−1). The validation results show that results obtained for one meat product is not always valid for other meat products. We were not able to obtain satisfactory results for N-nitrosohydroxyproline (NHPRO), N-nitrosodibenzylamine (NDBzA) and N-nitrosodiphenylamine (NDPhA). Application of the APCI interface improved the sensitivity of the method, because of less matrix interference, and gave the method a wider scope, as some NAs were ionisable only by APCI. However, it was only possible to ionize N-nitroso-thiazolidine-4-carboxylic acid (NTCA) and N-nitroso-2-methyl-thiazolidine-4-carboxylic acid (NMTCA) by ESI. The validated method was applied for the analysis of processed meat products and contents of N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR), N-nitrosomethylaniline (NMA), N-nitrosoproline (NPRO), NTCA, and NMTCA were found in one or several nitrite cured meat products, whereas none were detected in non-nitrite cured bacon.

Near ultraviolet radiation-mediated reaction between N-nitrosoproline and DNA: Isolation and identification of two new adducts, (R)- and (S)-8-(2-pyrrolidyl)-2′-deoxyguanosine

Abstract To explore possible genotoxicity of N-nitrosoproline (NPRO), we investigated near-ultraviolet radiation (UVA)-mediated chemical reaction of NPRO with 2′-deoxyguanosine (dG). An acidic solution containing NPRO and dG was irradiated with UVA and products were analyzed by HPLC. In addition to the three known modified guanine nucleosides, i.e., 2′-deoxyxanthosine, 8-oxo-7,8-dihydro-2′-deoxyguanosine and 2′-deoxyoxanosine, we found two products. These new products were isolated and identified as (R)- and (S)-8-(2-pyrrolidyl)-2′-deoxyguanosine (G1 and G2, respectively). Experiments using monochromatic UVA in the range 300–400 nm were performed to determine the relation between products-yield and wavelength, and the highest yields of both G1 and G2 were found to occur at 340 nm. This wavelength coincided with the absorption maximum of NPRO. We propose that the NO radical produced from NPRO by photolysis triggers the formation of these products. Further investigation using calf thymus DNA showed that G1 and/or G2, probably both, could be produced by UVA irradiation of DNA + NPRO as well.

A gas chromatography–mass spectrometry method for the quantitation of N-nitrosoproline and N-acetyl- S-allylcysteine in human urine: Application to a study of the effects of garlic consumption on nitrosation

Biomarkers in urine can provide useful information about the bioactivation of chemical carcinogens and can be used to investigate the chemoprotective properties of dietary nutrients. N-Nitrosoproline (NPRO) excretion has been used as an index for endogenous nitrosation. In vitro and animal studies have reported that compounds in garlic may suppress nitrosation and inhibit carcinogenesis. We present a new method for extraction and sensitive detection of both NPRO and N-acetyl- S-allylcysteine from urine. The latter is a metabolite of S-allylcysteine, which is found in garlic. Urine was acidified and the organic acids were extracted by reversed-phase extraction (RP–SPE) and use of a polymeric weak anion exchange (WAX–SPE) resin. NPRO was quantified by isotope dilution gas chromatography–mass spectrometry (GC–MS) using [ 13C 5]NPRO and N-nitrosopipecolinic acid (NPIC) as internal standards. This method was used to analyze urine samples from a study that was designed to test whether garlic supplementation inhibits NPRO synthesis. Using this method, 2.4 to 46.0 ng NPRO/ml urine was detected. The method is straightforward and reliable, and it can be performed with readily available GC–MS instruments. N-Acetyl- S-allylcysteine was quantified in the same fraction and detectable at levels of 4.1 to 176.4 ng/ml urine. The results suggest that 3 to 5 g of garlic supplements inhibited NPRO synthesis to an extent similar to a 0.5-g dose of ascorbic acid or a commercial supplement of aged garlic extract. Urinary NPRO concentration was inversely associated with the N-acetyl- S-allylcysteine concentration. It is possible that allyl sulfur compounds found in garlic may inhibit nitrosation in humans.

Effects of garlic ingestion in humans on the formation of urinary nitrosoproline

In vitro and animal studies suggest that compounds found in garlic may play a role in mediating the bioactivation of some carcinogens. N-nitrosoproline (NPRO) has been used in feeding studies as a biomarker for the formation of N-alkylnitrosamines. A controlled-feeding study was performed at Penn State University to test the hypothesis that garlic consumption inhibits nitrosamine formation. In this study, subjects (3 to 5 per group) consumed either 0 (control) 1, 3 or 5 grams of fresh garlic, 3 grams Kyolic (a commercial garlic supplement) or 500 mg vitamin C for 1 day following a 3 day run-in period in which 300 mg nitrate and 500 mg proline was consumed daily with a standard meal. Urine was collected during the last 24 hours and analyzed for NPRO. Aliquots were diluted with mobile phase, fractionated by isocratic reversed phase HPLC, and the fraction containing NPRO was dried and derivatized with pentafluorobenzyl bromide (PFB). The NPRO-PFB derivative was isolated using a second, gradient reversed-phase HPLC step and quantified by gas chromatography/ negative chemical ionization mass spectrometry. 13C5NPRO was used as the internal standard. Preliminary analysis of the samples indicates that NPRO excretion was reduced to nearly undetectable levels by treatments with 5 grams garlic, 3 grams Kyolic, and 500 mg vitamin C compared to controls. Analysis of the remaining samples is underway. In conclusion, the consumption of a single dose of prepared garlic, either fresh or as a supplement, reduced the excretion of NPRO under controlled-feeding conditions as much as a vitamin C supplement.

Determination of Total N-Nitroso Compounds by Chemical Denitrosation Using CuCl

A method for the determination of total N-nitroso compounds (NOC) by chemical denitrosation and subsequent chemiluminescence detection of evolved NO is described. Denitrosation was accomplished with CuCl in HCl at 70 degrees C. The detection limit for N-nitrosoproline (NPRO) was 1 pmol. NO formation from NPRO was linear (R(2) = 0.999) from 4 pmol to 2 nmol. Among the possible interfering compounds tested, only S-nitroso compounds contribute any significant interference. This method had several advantages over other similar methods: (1) A commercially available one-piece reaction vessel and a NO analyzer with software were used. (2) NO release occurred rapidly and was easily measured and quantified. (3) Compared to HBr or HI, CuCl was more convenient to work with and safe. (4) CuCl was suitable for samples in aqueous and most organic solvents. The application of this method to food, personal care products, and human body fluids demonstrates its utility.

Mutation, DNA strand cleavage and nitric oxide formation caused by N-nitrosoproline with sunlight: a possible mechanism of UVA carcinogenicity.

N-Nitrosoproline (NPRO) is endogenously formed from proline and nitrite. NPRO has been reported to be nonmutagenic and noncarcinogenic. In this study, we have detected the direct mutagenicity of NPRO plus natural sunlight towards Salmonella typhimurium. Furthermore, formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a mutagenic lesion, was observed in calf thymus DNA treated with NPRO plus simulated sunlight. The treatment with NPRO and sunlight induced single strand breaks in the superhelical replicative form of phage M13mp2 DNA. Single-strand DNA breaks also occurred in the human fibroblast cells on treatment with NPRO plus UVA, as detected by the comet assay. An analysis using scavengers suggested that both reactive oxygen species and NO radical mediate the strand breaks. The formation of nitric oxide was observed in NPRO solution irradiated with UVA. We analyzed the photodynamic spectrum of mutation induction and DNA breakage using monochromatic radiation at a series of wavelengths between 300 and 400 nm. Both mutation frequencies and DNA breakage were highest at the absorption maximum of NPRO, 340 nm. The co-mutagenic and co-toxic actions of NPRO and sunlight merit attention as possible mechanisms increasing the carcinogenic risk from UVA irradiation.

Hunting for electrophiles that harm human DNA: Frits Sobels Award Lecture.

This lecture is dedicated to Frits Sobels and his farsighted vision on research directions in genetic toxicology. Some accomplishments by the author's research group in the area of cancer etiology research and pre-clinical drug safety evaluation are presented. Praziquantel, an antischistosomal drug, was found to be devoid of any genetic effects which determined the drug companies to proceed with further safety evaluation and marketing. This highly efficient life-saving drug is now in use world wide. Biomonitoring methods have been developed to quantitate carcinogens, their metabolites or DNA adducts in humans exposed environmentally and endogenously to genotoxic agents. The methods were applied in ecological and case-control studies aimed at establishing causal relationships between exposure and disease. Results from both field studies in Iran and laboratory investigations supported the hypothesis that opium use, in particular ingestion of its pyrolysates, may be a risk factor for esophageal cancer in this region, probably acting together with nutritional deficiencies and thermal injury. By applying the nitrosoproline (NPRO) test in ecological studies on esophageal cancer causation in China some support was obtained for the involvement of N-nitroso compounds. In inhabitants of high risk areas endogenous nitrosamine synthesis could be markedly reduced by ingestion of vitamin C. Ultrasensitive detection methods for etheno-DNA adducts, which are formed by lipid peroxidation products resulting from increased oxidative stress, have been developed. Known cancer risk factors such as metal storage, chronic inflammatory processes and a high omega-6 PUFA fat diet increased the background level of these miscoding DNA adducts many times. They were found to increase progressively in premalignant lesions of cancer-prone tissues of humans and rodents, probably contributing to the genetic instability that drives cells to malignancy. Etheno-DNA adducts are thus promising markers to verify the efficiency of chemopreventive measures in humans.

Geographic association between urinary excretion of N‐nitroso compounds and oesophageal cancer mortality in china

Overnight urine samples were collected from approximately 60 male adults in each of 69 counties of China in 1989. Two specimens were collected from each subject--one after a loading dose of proline and ascorbic acid and another after a loading dose of proline only. Levels of N-nitrosamino acids and nitrate were measured in urine samples and correlated with cumulative mortality rates for subjects aged between 0 and 64 years in the 1970s. Oesophageal cancer mortality rates were positively and significantly associated with (i) urinary levels of excreted N-nitrosoproline (NPRO) (after proline and ascorbic acid loading or proline loading only), (ii) N-nitrososarcosine levels, and (iii) nitrosation potential (the decrease in the amount of urinary NPRO after adding ascorbic acid to the proline load). There were also positive correlations between the urinary level of NPRO or other N-nitrosamino acids and that of nitrate. The urinary excretion of nitrate was associated with consumption of various nitrate-rich vegetables. The results suggest that N-nitroso compounds (NOC) or other nitrite-derived carcinogens are implicated in the aetiology of oesophageal cancer in China.

Effects of fruit juices, processed vegetable juice, orange peel and green tea on endogenous formation of N-nitrosoproline in subjects from a high-risk area for gastric cancer in Moping County, China.

The effects of four fruit juices, processed vegetable juice, orange peel, green tea and low dose vitamin C on endogenous N-nitrosation in 86 subjects from a high-risk area for gastric cancer in Moping County, China were studied using urinary excretion of N-nitrosoproline (NPRO) as an indicator. After ingestion of 300 mg L-proline, urinary excretion of NPRO was significantly increased from a baseline of 2.5 +/- 1.6 micrograms/day to 8.7 +/- 6.2 micrograms/day. (P < 0.001). Vitamin C (75 mg) administration significantly reduced NPRO formation (62.3%, P < 0.002) although NPRO excretion remained higher than the baseline level (4.2 +/- 1.3 vs 2.2 +/- 1.2 micrograms/day, P < 0.001). Intake of fruit juices and green tea extracts (containing 75 mg vitamin C) or of orange peel powder (containing 3 mg vitamin C) together with 300 mg L-proline inhibited NPRO formation effectively to the baseline level or to levels significantly lower than the baseline level (P < 0.05-0.005). A processed juice of a number of vegetables (300 ml) significantly catalysed endogenous nitrosation (14.7 +/- 11.8 vs 9.4 +/- 4.7 micrograms/day, P < 0.05). Endogenous N-nitrosation was unaffected by the presence of intragastric lesions. The present study shows that endogenous nitrosation in this population is profoundly affected by environmental factors and that inhibitors, such as vitamin C, alpha-tocopherol and other non-nutritive compounds in the foods do inhibit endogenous nitrosation either synergistically or in an additive manner. The significance of fruits and vegetables in prevention of human cancers is discussed.

Studies on endogenous formation of N-nitroso compounds in the guinea pig supplemented with proline or thioproline and sodium nitrate

The endogenous formation of N-nitrosoproline (NPRO) and N-nitrosothioproline (NTPRO, N-nitrosothiazolidine-4-carboxylic acid) was studied by monitoring their excretion in the urine of guinea pigs given oral doses of 10 mg proline or thioproline after supplementation with 34 mg (0.4 mmol) sodium nitrate. In order to estimate the conversion of nitrate to nitrate, the animals were also supplemented with 3.5 mg (0.05 mmol) sodium nitrate instead of sodium nitrate. In animals fed commercial diets, the excretion of NPRO and NTPRO under supplementation with sodium nitrate was 2.0 μg and 28.7 μg/animal/day, respectively, whereas the excretion under supplementation with sodium nitrate was 0.7 μg and 13.3 μg/animal/day, respectively. The higher excretion of NTPRO than NPRO in each case shows that thioproline is more effective for nitrite trapping than proline. The animals supplemented with nitrate excreted more than twice the amounts of NPRO or NTPRO than those supplemented with nitrite. It is assumed, therefore, that more than 0.1 mmol nitrate is reduced to nitrite and takes part in the endogenous nitrosation of the guinea pig. When various concentrations of l-ascorbic acid (AsA), known to inhibit the formation of N-nitroso compounds, were also administered orally to animals immediately after supplementation with sodium nitrate, the NPRO excretion decreased with increasing AsA concentration. These data indicate that the guinea pig, which is unable to synthesize AsA as well as humans, may be an appropriate animal model for evaluation of the endogenous nitrosation ability of humans ingesting nitrate.

Teas and tea components as inhibitors of carcinogen formation in model systems and man

Background. Epidemiological evidence points to a cancer protective role of green-yellow-orange vegetables and fruits. The involvement of teas as a protective factor in carcinogenesis has not received the attention it seems to merit. To gain relevant information, attempts were made to stimulate in vitro those conditions to which human groups are actually exposed. Methods. The inhibitory effects of infusions of Chinese, Japanese, and Ceylonese teas were examined by adding them to a nitrosation mixture consisting of 0.8 mg sodium nitrite and 340 mg equivalent of a widely consumed salt-preserved fish (Pak Wik) and estimating the frequency of mutants in TA 1535 strain of Salmonella typhimurium. Results. The tea samples exhibited a strong inhibitory effect at concentrations that are actually ingested by man. A comparable inhibition was obtained by several tea phenolics. A second series of experiments dealt with the formation of nitrosoproline (NPRO) which can be strongly inhibited in vitro by the tea infusions and tea phenolics. The effects of the tea infusions and caffeic acid on the endogeneous formation of NPRO in man were examined by having volunteers ingest 300 mg sodium nitrate and 30 min later 300 mg proline, collecting urine samples over a 24-hr period, and estimating the excreted NPRO. The tested teas, at doses regularly consumed, again exerted a strong inhibitory effect on endogeneous NPRO formation in humans. Comparable inhibitory effects were obtained by ingesting caffeic acid, chlorogenic acid, or ferulic acid with the nitrosation mixture. Conclusions. These results indicate that the simultaneous intake of teas with food products that are being nitrosated within the stomach of human subjects should exert a protective, beneficial effect.

Influence of fruit and vegetable juices on the endogenous formation of N-nitrosoproline and N-nitrosothiazolidine-4-carboxylic acid in humans on controlled diets.

Water, green pepper, pineapple, tomato, strawberry, carrot, and celery juices were made 46 mg/100 ml in ascorbic acid by the addition of distilled water or ascorbate. The ability of each juice to inhibit endogenous formation of N-nitrosoamino acids (NAA) in humans was determined in controlled experiments. Sixteen men consumed a standard diet low in nitrate and ascorbic acid for 18 consecutive days. Nitrate (5.24 mmol) and L-proline (4.35 mmol) were given orally on days 3, 5, 7, 9, 11, 13, 15, 17 and 18. On days 3 and 18, L-proline was immediately followed by 100 ml distilled water (positive control). On days 5, 7, 9, 11, 13, 15 and 17, L-proline was immediately followed by 100 ml juice or 46 mg ascorbate in 100 ml distilled water (treatment). Only diet was given in between dosing days to ensure baseline levels of NAA excretion. Urine was collected for 24 h following treatments and analyzed for NAA. Green pepper, pineapple, tomato, strawberry and carrot treatments significantly inhibited N-nitrosoproline (NPRO) formation relative to the positive control. Also, green pepper, pineapple and tomato juices significantly inhibited NPRO formation relative to ascorbic acid alone. Green pepper significantly inhibited N-nitrosothiazolidine-carboxylic acid formation relative to ascorbic acid alone. These data demonstrate that green pepper, tomato, pineapple, strawberry and carrot juice have greater ability to inhibit endogenous nitrosation than would be expected based solely on their ascorbate content.

InternationalN‐nitroso compounds check sample programme: Report on the performance in the second study dedicated to their determination in beer and malt

The second check sample survey for the determination of N-nitrosamines in beer and malt has been initiated. Each laboratory received four samples: two beer, from the same batch, spiked respectively with 0.5 microgram/l N-nitrosodimethylamine (NDMA) and 4 micrograms/l NDMA plus 30 micrograms/l N-nitrosoproline (NPRO) and two naturally contaminated malts. Sixteen laboratories sent their results and two apologized in view of analytical problems. Results from this have been statistically evaluated. Comparison to those from the previous study demonstrated a distinct improvement in the results from the analysis of NDMA in beer and malt. Little has been achieved for the other two common contaminants of beer and malt, N-nitrosopyrrolidine (NPYR) and NPRO. For this latter compound in beer, however, some hope came from the data produced by the method of Sen et al. (1983), but the number of results is insufficient to allow firm conclusions to be drawn.