Food Additives & Contaminants
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
Effect of dietary fats on endogenous formation of N‐nitrosamines from nitrate in germ‐free and conventional rats and rats harbouring a human flora F. W. Ward , M. E. Coates , C. B. Cole & R. Fuller To cite this article: F. W. Ward , M. E. Coates , C. B. Cole & R. Fuller (1990) Effect of dietary fats on endogenous formation of N‐nitrosamines from nitrate in germ‐free and conventional rats and rats harbouring a human flora, Food Additives & Contaminants, 7:5, 597-604, DOI: 10.1080/02652039009373925 To link to this article: http://dx.doi.org/10.1080/02652039009373925
Published online: 10 Jan 2009.
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Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac20 Download by: [Universite Laval]
Date: 13 November 2015, At: 08:07
FOOD ADDITIVES AND CONTAMINANTS, 1990, VOL. 7, NO. 5, 5 9 7 - 6 0 4
Effect of dietary fats on endogenous formation of N-nitrosamines from nitrate in germ-free and conventional rats and rats harbouring a human flora F. W. WARD†, M. E. COATES†‡, C. B. COLE§ and R. FULLER§ † Robens Institute, University of Surrey, Guildford, Surrey GU2 5XH, UK; § AFRC Food Research Institute, Shinfield, Reading RG2 9AT, UK
Food Additives & Contaminants 1990.7:597-604.
(Received 19 December 1989; revised 20 February 1990; accepted 21 February 1990) Urinary excretion of N-nitrosoproline (NPRO) was measured in groups of four germ-free (GF) and conventional (CV) rats given a purified diet with or without inclusion of 100 g butterfat, coconut oil or maize oil/kg, and drinking water containing 0.235 M-NaNO3. In the CV environment rats given the fat-supplemented diets excreted significantly less NPRO than those on the low-fat diet. No corresponding decrease in NPRO excretion occurred in the GF environment. Nitrate reductase activity was measured in stomach contents and homogenates of stomach tissue from GF and CV rats given the different diets. No activity was detected in any of the contents from GF rats. Nitrate reductase activity was significantly reduced in contents from all the CV rats given the fat-supplemented diets, the effect being most marked in those given butterfat. Activity was much lower in tissue homogenates from GF rats than in those from their CV counterparts, but was not affected by diet in either environment. Groups of four CV rats, or rats harbouring a human faecal flora (HF), were given the purified diet with or without addition of 100 g butterfat or maize oil/kg and drinking water containing 0.235 M-NaNO3. All groups given the fatsupplemented diets excreted significantly less NPRO than the corresponding groups on the low-fat diet with the exception of the HF rats given butterfat. It is concluded that the reduced excretion of NPRO by rats given diets containing fat was mainly due to inhibition of microbial nitrate reductase activity in the foregut. The smaller effect of butterfat on the HF rats accords with earlier findings in human subjects. Keywords: Fat, nitrosamines, gut flora, nitrate reductase
Introduction
The potential toxicity of nitrate depends on its reduction to nitrite, which may subsequently react with amines or amides from the food to produce carcinogenic N-nitrosamines. The existence both of mammalian and microbial nitrate reductase systems has been demonstrated in the gastrointestinal tract of the rat (Ward et al. 1986). Nitrosation proceeds non-enzymatically at and below pH 4 and occurs mainly in the stomach. Urinary excretion of 7V-nitrosoproline (NPRO), a stable Nnitrosated compound, is considered to be a valid index of endogenous Nnitrosation (Ohshima et al. 1982). In a previous investigation (Ward and Coates, 1987a) we showed that inclusion of 100 g fat/kg in a purified diet for rats significantly reduced their urinary excretion of NPRO, and that butterfat had a markedly greater effect than several vegetable ‡ Present address: Department of Biochemistry, University of Surrey, UK. 0265-203X/90 $3.00 © Taylor & Francis Ltd
598
F. W. Ward et al.
Food Additives & Contaminants 1990.7:597-604.
oils. Since the presence of fat in the diet did not affect absorption of NPRO, or the intragastric iV-nitrosation reaction, it was postulated that the effect was due to inhibition of nitrate reductase activity. Nitrate reductase activity in the stomach contents from conventional (CV) rats was apparently of microbial origin as none was detected in stomach contents from their germ-free (GF) counterparts. The activity in stomach contents from CV rats given a low-fat diet was significantly reduced when butterfat was included in the incubation medium. The suppressive effect of dietary fat on nitrosamine excretion was also shown to occur in man, but butterfat was not more effective than vegetable oil (Ward et al. 1988). The difference in response between rat and man might be the result of species differences in composition of the gut microfloras. The studies now reported were made in GF and CV rats to confirm that the suppressive effect of fat on nitrosamine excretion was mediated through the microflora. To obtain evidence on possible species differences, comparisons were made between CV rats and rats harbouring a human faecal flora.
Materials and methods Animals and diets Experiments were done on male and female Lister hooded rats 7-9 weeks old, bred on the premises. GF and CV rats were maintained as previously described (Ward et al. 1986, Ward and Coates 1987a). They were fed on purified diets with or without 100 g fat/kg (Ward and Coates 1987a). Since the inclusion of fat increased the energy density of the diet the amounts of the other ingredients in the fatsupplemented diets were adjusted to be in the same proportion relative to energy as in the low-fat diet. Previous analyses of the dietary ingredients and drinking water showed no traces of nitrite or nitrosamines. Diets both for CV and GF rats were sterilized by gamma-radiation at 50 kGy. Analytical methods Nitrite determination. Nitrite was determined in solution by the method of Shechter et al. (1972) using iV-1-naphthylethylene diamine dihydrochloride (Sigma Chemical Co. Ltd. Poole, Dorset) as the coupling reagent and the absorbance read at 543 nm. ^-nitrosamine determination. Af-nitrosoproline (NPRO) was analysed on a Pye 104 gas chromatograph coupled to a thermal energy analyser (Thermoelectron Corp., Mass., USA) using the modified method of Ohshima (1983) as described by Ward et al. (1986). Acidified aqueous samples were extracted with ethyl acetate on Extralut columns (E. Merck, Darmstadt, FRG). Following concentration on a rotary evaporator at 50°C an ethereal solution of diazomethane was used to methylate the extracts. Separation was achieved on a glass column packed with 10% Carbowax 20 M on Chromosorb W-HP, 100-120 mesh, and heated to 185°C. Measurement of nitrate reductase activity. Nitrate reductase activity was measured in homogenates of stomach tissue and in stomach contents. The samples were homogenized in cold sterile Krebs Ringer phosphate (KRP) buffer, pH 7-4,
Dietary fats and NPRO excretion in rats
599
containing 0-235 M-sodium nitrate and incubated at 37°C for 24 h. The nitrite formed was determined as described above. Preparation of samples. The animals were killed by cervical dislocation and the abdomen was exposed. Stomach contents were suspended in 10 ml KRP buffer. An homogenizer was not available inside the isolators so the washed stomach tissue from both CV and GF rats was ground by hand in a mortar containing sterile sand, 40-100 mesh (BDH Chemicals Ltd, Poole, Dorset), and 15 ml KRP buffer. All GF samples were prepared within the isolator and sealed into incubation bottles before removal.
Food Additives & Contaminants 1990.7:597-604.
Statistical analysis Student's /-test was used to calculate the significance of difference between means of two treatments, and analysis of variance was done on the results of experiments with more than two treatments. Experimental procedure
Experiment 1 This experiment in vivo compared the effect of dietary fats on urinary excretion of NPRO in the presence and absence of a gut microflora. Groups of two male and two female GF rats and their CV counterparts were fed for 3 weeks on a low-fat diet, or one supplemented with 100 g butterfat, coconut oil or maize oil/kg. Their drinking water contained 0-235 M sodium nitrate. Urine was collected daily in 5 ml 1 M-NaOH over the final 7 days and a portion analysed for NPRO. Experiment 2 This was an investigation of the effects of dietary fats on nitrate reductase activity in stomach contents and tissue in the presence and absence of a gut microflora. Groups of two male and two female GF rats and their CV counterparts ate the diets described above and drank 0-235 M sodium nitrate for 2 weeks. After an overnight fast for 18 h they were allowed access to food for 1 h. They were then killed and the stomach contents and tissue suspended in 10 ml and 15 ml of KRP buffer respectively. Nitrate reductase activity was determined in each. Experiment 3 This was a comparison of the influence of dietary fat on NPRO excretion by CV rats and those harbouring a human faecal flora. GF rats were inoculated by mouth with ca. 1 ml of a 20% w/v suspension of faeces from a healthy adult male. The inoculum was equivalent to approximately 5 X 1010 microorganisms per rat. Groups of two males and two females were fed for 3 weeks on the low-fat diet supplemented with 100 g butterfat or maize oil/kg, with 0-235 MNaNO3 in the drinking water. Urine was collected and analysed as described in experiment 1. Results
In experiments 1 and 2 the GF rats remained uncontaminated. Experiment 1 NPRO excretion was significantly less by CV rats fed on the fat-supplemented
600
F. W. Ward et al. Table 1. Urinary excretion of TV-nitrosoproline (NPRO) by germ-free (GF) and conventional (CV) rats fed on diets with or without addition of 100 g fat/kg and 0-235 M-sodium nitrate in drinking water for 3 weeks (mean values with their standard errors for four rats). NPRO excreted CV
Food Additives & Contaminants 1990.7:597-604.
GF
Diet
Mean (ng/day)
Low fat Butterfat Coconut oil Maize oil
198** 210* 178 267*
SE
Mean (ng/day)
SE
±35 ±28 ±54 ±21
303 137*** 161*'* 175***
±25 ±12 ±11 ±11
Least significant difference at P
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
Effect of dietary fats on endogenous formation of N‐nitrosamines from nitrate in germ‐free and conventional rats and rats harbouring a human flora F. W. Ward , M. E. Coates , C. B. Cole & R. Fuller To cite this article: F. W. Ward , M. E. Coates , C. B. Cole & R. Fuller (1990) Effect of dietary fats on endogenous formation of N‐nitrosamines from nitrate in germ‐free and conventional rats and rats harbouring a human flora, Food Additives & Contaminants, 7:5, 597-604, DOI: 10.1080/02652039009373925 To link to this article: http://dx.doi.org/10.1080/02652039009373925
Published online: 10 Jan 2009.
Submit your article to this journal
Article views: 6
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac20 Download by: [Universite Laval]
Date: 13 November 2015, At: 08:07
FOOD ADDITIVES AND CONTAMINANTS, 1990, VOL. 7, NO. 5, 5 9 7 - 6 0 4
Effect of dietary fats on endogenous formation of N-nitrosamines from nitrate in germ-free and conventional rats and rats harbouring a human flora F. W. WARD†, M. E. COATES†‡, C. B. COLE§ and R. FULLER§ † Robens Institute, University of Surrey, Guildford, Surrey GU2 5XH, UK; § AFRC Food Research Institute, Shinfield, Reading RG2 9AT, UK
Food Additives & Contaminants 1990.7:597-604.
(Received 19 December 1989; revised 20 February 1990; accepted 21 February 1990) Urinary excretion of N-nitrosoproline (NPRO) was measured in groups of four germ-free (GF) and conventional (CV) rats given a purified diet with or without inclusion of 100 g butterfat, coconut oil or maize oil/kg, and drinking water containing 0.235 M-NaNO3. In the CV environment rats given the fat-supplemented diets excreted significantly less NPRO than those on the low-fat diet. No corresponding decrease in NPRO excretion occurred in the GF environment. Nitrate reductase activity was measured in stomach contents and homogenates of stomach tissue from GF and CV rats given the different diets. No activity was detected in any of the contents from GF rats. Nitrate reductase activity was significantly reduced in contents from all the CV rats given the fat-supplemented diets, the effect being most marked in those given butterfat. Activity was much lower in tissue homogenates from GF rats than in those from their CV counterparts, but was not affected by diet in either environment. Groups of four CV rats, or rats harbouring a human faecal flora (HF), were given the purified diet with or without addition of 100 g butterfat or maize oil/kg and drinking water containing 0.235 M-NaNO3. All groups given the fatsupplemented diets excreted significantly less NPRO than the corresponding groups on the low-fat diet with the exception of the HF rats given butterfat. It is concluded that the reduced excretion of NPRO by rats given diets containing fat was mainly due to inhibition of microbial nitrate reductase activity in the foregut. The smaller effect of butterfat on the HF rats accords with earlier findings in human subjects. Keywords: Fat, nitrosamines, gut flora, nitrate reductase
Introduction
The potential toxicity of nitrate depends on its reduction to nitrite, which may subsequently react with amines or amides from the food to produce carcinogenic N-nitrosamines. The existence both of mammalian and microbial nitrate reductase systems has been demonstrated in the gastrointestinal tract of the rat (Ward et al. 1986). Nitrosation proceeds non-enzymatically at and below pH 4 and occurs mainly in the stomach. Urinary excretion of 7V-nitrosoproline (NPRO), a stable Nnitrosated compound, is considered to be a valid index of endogenous Nnitrosation (Ohshima et al. 1982). In a previous investigation (Ward and Coates, 1987a) we showed that inclusion of 100 g fat/kg in a purified diet for rats significantly reduced their urinary excretion of NPRO, and that butterfat had a markedly greater effect than several vegetable ‡ Present address: Department of Biochemistry, University of Surrey, UK. 0265-203X/90 $3.00 © Taylor & Francis Ltd
598
F. W. Ward et al.
Food Additives & Contaminants 1990.7:597-604.
oils. Since the presence of fat in the diet did not affect absorption of NPRO, or the intragastric iV-nitrosation reaction, it was postulated that the effect was due to inhibition of nitrate reductase activity. Nitrate reductase activity in the stomach contents from conventional (CV) rats was apparently of microbial origin as none was detected in stomach contents from their germ-free (GF) counterparts. The activity in stomach contents from CV rats given a low-fat diet was significantly reduced when butterfat was included in the incubation medium. The suppressive effect of dietary fat on nitrosamine excretion was also shown to occur in man, but butterfat was not more effective than vegetable oil (Ward et al. 1988). The difference in response between rat and man might be the result of species differences in composition of the gut microfloras. The studies now reported were made in GF and CV rats to confirm that the suppressive effect of fat on nitrosamine excretion was mediated through the microflora. To obtain evidence on possible species differences, comparisons were made between CV rats and rats harbouring a human faecal flora.
Materials and methods Animals and diets Experiments were done on male and female Lister hooded rats 7-9 weeks old, bred on the premises. GF and CV rats were maintained as previously described (Ward et al. 1986, Ward and Coates 1987a). They were fed on purified diets with or without 100 g fat/kg (Ward and Coates 1987a). Since the inclusion of fat increased the energy density of the diet the amounts of the other ingredients in the fatsupplemented diets were adjusted to be in the same proportion relative to energy as in the low-fat diet. Previous analyses of the dietary ingredients and drinking water showed no traces of nitrite or nitrosamines. Diets both for CV and GF rats were sterilized by gamma-radiation at 50 kGy. Analytical methods Nitrite determination. Nitrite was determined in solution by the method of Shechter et al. (1972) using iV-1-naphthylethylene diamine dihydrochloride (Sigma Chemical Co. Ltd. Poole, Dorset) as the coupling reagent and the absorbance read at 543 nm. ^-nitrosamine determination. Af-nitrosoproline (NPRO) was analysed on a Pye 104 gas chromatograph coupled to a thermal energy analyser (Thermoelectron Corp., Mass., USA) using the modified method of Ohshima (1983) as described by Ward et al. (1986). Acidified aqueous samples were extracted with ethyl acetate on Extralut columns (E. Merck, Darmstadt, FRG). Following concentration on a rotary evaporator at 50°C an ethereal solution of diazomethane was used to methylate the extracts. Separation was achieved on a glass column packed with 10% Carbowax 20 M on Chromosorb W-HP, 100-120 mesh, and heated to 185°C. Measurement of nitrate reductase activity. Nitrate reductase activity was measured in homogenates of stomach tissue and in stomach contents. The samples were homogenized in cold sterile Krebs Ringer phosphate (KRP) buffer, pH 7-4,
Dietary fats and NPRO excretion in rats
599
containing 0-235 M-sodium nitrate and incubated at 37°C for 24 h. The nitrite formed was determined as described above. Preparation of samples. The animals were killed by cervical dislocation and the abdomen was exposed. Stomach contents were suspended in 10 ml KRP buffer. An homogenizer was not available inside the isolators so the washed stomach tissue from both CV and GF rats was ground by hand in a mortar containing sterile sand, 40-100 mesh (BDH Chemicals Ltd, Poole, Dorset), and 15 ml KRP buffer. All GF samples were prepared within the isolator and sealed into incubation bottles before removal.
Food Additives & Contaminants 1990.7:597-604.
Statistical analysis Student's /-test was used to calculate the significance of difference between means of two treatments, and analysis of variance was done on the results of experiments with more than two treatments. Experimental procedure
Experiment 1 This experiment in vivo compared the effect of dietary fats on urinary excretion of NPRO in the presence and absence of a gut microflora. Groups of two male and two female GF rats and their CV counterparts were fed for 3 weeks on a low-fat diet, or one supplemented with 100 g butterfat, coconut oil or maize oil/kg. Their drinking water contained 0-235 M sodium nitrate. Urine was collected daily in 5 ml 1 M-NaOH over the final 7 days and a portion analysed for NPRO. Experiment 2 This was an investigation of the effects of dietary fats on nitrate reductase activity in stomach contents and tissue in the presence and absence of a gut microflora. Groups of two male and two female GF rats and their CV counterparts ate the diets described above and drank 0-235 M sodium nitrate for 2 weeks. After an overnight fast for 18 h they were allowed access to food for 1 h. They were then killed and the stomach contents and tissue suspended in 10 ml and 15 ml of KRP buffer respectively. Nitrate reductase activity was determined in each. Experiment 3 This was a comparison of the influence of dietary fat on NPRO excretion by CV rats and those harbouring a human faecal flora. GF rats were inoculated by mouth with ca. 1 ml of a 20% w/v suspension of faeces from a healthy adult male. The inoculum was equivalent to approximately 5 X 1010 microorganisms per rat. Groups of two males and two females were fed for 3 weeks on the low-fat diet supplemented with 100 g butterfat or maize oil/kg, with 0-235 MNaNO3 in the drinking water. Urine was collected and analysed as described in experiment 1. Results
In experiments 1 and 2 the GF rats remained uncontaminated. Experiment 1 NPRO excretion was significantly less by CV rats fed on the fat-supplemented
600
F. W. Ward et al. Table 1. Urinary excretion of TV-nitrosoproline (NPRO) by germ-free (GF) and conventional (CV) rats fed on diets with or without addition of 100 g fat/kg and 0-235 M-sodium nitrate in drinking water for 3 weeks (mean values with their standard errors for four rats). NPRO excreted CV
Food Additives & Contaminants 1990.7:597-604.
GF
Diet
Mean (ng/day)
Low fat Butterfat Coconut oil Maize oil
198** 210* 178 267*
SE
Mean (ng/day)
SE
±35 ±28 ±54 ±21
303 137*** 161*'* 175***
±25 ±12 ±11 ±11
Least significant difference at P
