Food Additives & Contaminants
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
Effects of harmane on growth and in vivo metabolism of aflatoxin B1 in male and female rats Catherine Billaud To cite this article: Catherine Billaud (1991) Effects of harmane on growth and in vivo metabolism of aflatoxin B1 in male and female rats, Food Additives & Contaminants, 8:6, 713-722, DOI: 10.1080/02652039109374029 To link to this article: http://dx.doi.org/10.1080/02652039109374029
Published online: 10 Jan 2009.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac20 Download by: [University of Florida]
Date: 08 November 2015, At: 07:39
FOOD ADDITIVES AND CONTAMINANTS, 1991, VOL. 8, NO. 6, 713-722
Effects of harmane on growth and in vivo metabolism of aflatoxin B1 in male and female rats CATHERINE BILLAUD Laboratoire de Biochimie Industrielle et Agro-alimentaire, Conservatoire National des Arts et Métiers, 292, rue Saint-Martin, 75141 Paris Cedex 03, France
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(Received 19 April 1991, revised 14 October 1991; accepted 11 November 1991) The role of harmane, a β-carboline formed during pyrolysis of tryptophan, on the metabolism of AFB1, growth and some parameters of the nutritional status was investigated in the rat. Male and female Wistar rats were fed a semi-synthetic diet containing AFB1 (2 ppm), harmane (250 ppm) or both compounds, for 33 days after weaning. Qualitative and quantitative differences in the urinary and faecal excretion of parental compound and metabolites were assessed by HPLC analysis. Harmane did not modify appreciably the growth and the other nutritional parameters studied. Similar excretion patterns of AFB1 metabolites were observed in males and females. Harmane caused a limited increase in the excretion of AFM1 in faeces but not in urine, without altering the growth process in rats of either sex. Keywords: Harmane, aflatoxin B1, metabolism, growth, rat Introduction
Aflatoxin Bi (AFBi) is a potent hepatotoxic and hepatocarcinogen for animals and possibly for man. Like many other toxins, AFBi requires metabolic activation to become oncogenic. Microsomal cytochrome(s) P450-linked monooxygenases are involved in the formation of highly reactive intermediates such as DNA-reactive epoxides (Essigman et al., 1982). These systems, in conjunction with cytosolic enzymes convert AFBi into various metabolites such as AFMi (hydroxylated form), AFPi (demethylated form) and aflatoxicol (Ro, reduced form). Some of these products may also be conjugated with glucuronic acid, sulphate or glutathione and excreted into bile, urine and faeces (Raj and Lotlikar, 1984). Both natural and synthetic compounds present in animal and human diets have been shown to reduce AFBi-induced carcinogenesis (Godlewski et al. 1985, Monroe et al. 1986, Wattenberg et al. 1986, Kensler et al. 1987, Bhattacharya and Firozi 1988, Goeger et al. 1988, Ramsdell and Eaton 1988, Fong and Bailey 1990). They could favour the formation of less carcinogenic metabolites such as AFMi and/or other readily extractable hydrophilic metabolites (Fukayama and Hsieh 1985). Contrastingly, numerous chemicals present in minute amounts in food are generated by food processing and may act as initiators of carcinogenesis. In this view, the cooking of protein-rich food and more particularly the pyrolysis of proteins and indole derivatives generate heterocyclic amines exhibiting mutagenic activity higher than AFBi and a carcinogenic potency, when given orally to rodents (Matsukura et al. 1981, Takayama et al. 1984, Sugimura 1988). 0265-203X/91 $3.00 © 1991 Taylor & Francis Ltd.
714
Catherine Billaud
o H H
R
1
R= H
X= H
NORHARMANE
2
R = CH 3
X= H
HARMANE
Downloaded by [University of Florida] at 07:39 08 November 2015
Figure 1. Structure of two 0-carbolines, harmane and norharmane.
The (3-carbolines harmane and norharmane devoid of exocyclic amino groups (figure 1), though not mutagenic per se, are known to enhance the genotoxicity of various chemicals (Nagao et al. 1977, Gupta et al. 1989). These compounds are found in various beverages and foodstuffs at levels ranging from 0-1 to 1000 ng/g (Adachi et al. 1991) and represent 8-10% by weight of the pyrolysis product of DL-tryptophan. In a previous experiment (unpublished results) we noticed that tryptophan pyrolysate when administered to rats at the dose of 2 g per kg of diet (i.e. 200 ppm approx. of harmane + norharmane), partly inhibited the hepatocarcinogenesis induced by AFBi. Moreover, in rats fed the tryptophan pyrolysate-enriched diet, we observed a decrease in the urinary excretion of AFMi, one of the major metabolites of AFBi. These effects were more pronounced in males than in females. In the present work, the effects of harmane on growth of male and female rats fed an AFBi-containing diet were studied. An attempt was also made to determine the identity and excretion level of some AFBi metabolites. Materials and methods
Animals Weaning Wistar CF rats of both sexes averaging 46 g body weight were purchased from R. Janvier breeding laboratory (Le Genest St Isle, France) and were divided into groups of 12 animals per treatment and per sex. Series of three or four rats were kept in stainless steel cages, males separated from females and fed ad libitum one of the four experimental diets. Chemicals and special diets The semi-synthetic basal diet used was prepared weekly in our laboratory; its composition is listed in table 1. In the experimental diets, a concentrated ethanolic solution of harmane (H) and/or aflatoxin Bi (AFBi) was thoroughly blended with the balanced mixture to provide a final concentration of 2 mg aflatoxin Bi and 250 mg harmane per kg of diet. The same amount of ethanol was incorporated to the control diet. Aflatoxin Bi (crystalline form) was purchased from Makor Chemicals (Jerusalem, Israel); harmane (l-methyl-9H-pyrido[3,4-b]indole) free base (crystalline form) came from Sigma Chemicals Company (St Louis, MO). Experimental design During the study, animals were divided into eight experimental groups as
Effects of harmane on growth and AFBi metabolism
715
Table 1. Composition of basal diet in control and experimental groups (identical for male and female rats) (g/kg).
Downloaded by [University of Florida] at 07:39 08 November 2015
Lactic casein Soyamin 90 Cellulose Cornstarch Vitamin mixture 8 Mineral mixture b Lard Edible soybean oil (ml) Methionine Choline 40 per 100 (ml) Sucrose q.s.
20 130 20 200 10 40 100 60 1 2-5 1000
a Vitamin mixture contained per kg: Vitamin A (retinol palmitate IU, 250000/g) 1-2 g; Vitamin D 2 or D 3 (IU 500000/g), 0-3 g; Vitamin E (DL-a-tocopherol succinic acid) (IU 1200/g), 3-3 g; Vitamin K3 (menadione), 300 mg; Vitamin Bi (thiamine HC1), 150 mg; Vitamin B 2 (riboflavin), 250 mg; vitamin PP or B3 (niacin) 3000 mg; vitamin B5 (calcium pantothenate) 500 mg; vitamin B6 (pyridoxine HC1) 150 mg; vitamin B7 (inositol), 10000 mg; vitamin H or Bg (D-biotin) 16 mg; vitamin Bc or B 9 (folic acid), 50 mg; vitamin B12 (cyanocobalamin), 50 mg; paraminobenzoic acid, 5000 mg; cornstarch q.s. 1000 g b L . Gueguen salt mixture (INRA, La Miniere: CM 5420).
follows: —two groups C (control): male or female rats given the plain semi-synthetic diet without chemical added throughout the experiment; —two groups H (harmane): male or female rats given the diet with harmane at the level of 250 ppm; —two groups AFBi (aflatoxin Bi): male or female rats given the diet with AFBt at the level of 2 ppm; —two groups AFBi + H (aflatoxin B t + harmane): male or female rats given the diet with AFBi plus harmane at the above mentioned concentrations. After a period of 28 days, male and female rats were randomly allocated to individual metabolism cages for 5 days. We used seven rats from C and H group and 12 from AFBi and AFBi + H. Food containers were weighed daily in order to evaluate the uptake of food and contaminants. Their body weight was recorded at the beginning and at the end of the metabolism period. Individual faecal and urine excretions were collected daily, weight and volumes recorded. When appropriated, gross observations of appearance and behaviour were noticed. Sampling of urine and faeces Urine samples from 5 days were collected free of faecal contamination into graduated vessels containing a few ml of toluene in order to protect chemical compounds from oxidation, thereafter specimens were each filtered through glass wool, then transferred to flasks and adjusted to a final 100 ml with distilled water.
716
Catherine Billaud
Downloaded by [University of Florida] at 07:39 08 November 2015
Pooled faeces from 5 days were dried in a lyophilizator and finely ground with aid of a mini ballgrinder. Both samples were kept frozen until determination of aflatoxins levels. Material Liquid chromatography apparatus. Reversed-phase chromatography was performed using Constametric IIG HPLC system equipped with Constametric I and II pumps (LDC Milton Roy), a model UHP - 7 K Valco injector (25 /il sample loop) and a gradient master solvent programmer (LDC). The aflatoxins were detected with a model 1311 Fluoromonitor III, fluorometric detector (LDC) equipped with an excitation filter (340-380 nm transmission), and emission filter (>417nm transmission) and a 30 /tl flow cell. Peak areas were calculated with a LDC 304 - 50 computing integrator. Separation of the aflatoxins was achieved on a 25 cmx4-6 mm i.d., 5 urn, C18 column (Vydac 201 TP 54, Hesperia, California), operating at 1-1 ml/min (nominal pressure of =2000 psi). Reagents. Reagent grade acetone, chloroform, dichloromethane, «-hexane, ethanol, benzene, diethylether and anhydrous sodium sulphate, HPLC grade acetonitrile were from Prolabo (Paris, France). For HPLC elution system solvent, analytical reagent methanol (Prolabo, France) was redistilled in our laboratory. HPLC elution solvent consisted of a water/methanol/acetonitrile mixture (70:15:15). The mixture was degassed by sonication just prior to analysis. Standard solutions. Aflatoxins were obtained from Makor Chemicals Ltd (Jerusalem, Israel), AFMi and AFBi from Aspergillus Jlavus, AFPi chemically derived from Aspergillus Jlavus and aflatoxicol I ( = Ro), natural isomer. Stock solutions of each aflatoxin were prepared in benzene/acetonitrile (9 + 1), and kept at -20°C in the dark. Mixed aflatoxin working solutions were diluted with methanol to provide appropriate concentrations. Extraction procedure Aliquots of urine (10 ml) were extracted twice, each time with 50 ml volumes of chloroform/acetone (1:1) and dried over 30 g anhydrous sodium sulphate. After evaporation of organic solvents using a rotavapor (Bu'chi) at a temperature
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
Effects of harmane on growth and in vivo metabolism of aflatoxin B1 in male and female rats Catherine Billaud To cite this article: Catherine Billaud (1991) Effects of harmane on growth and in vivo metabolism of aflatoxin B1 in male and female rats, Food Additives & Contaminants, 8:6, 713-722, DOI: 10.1080/02652039109374029 To link to this article: http://dx.doi.org/10.1080/02652039109374029
Published online: 10 Jan 2009.
Submit your article to this journal
Article views: 5
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac20 Download by: [University of Florida]
Date: 08 November 2015, At: 07:39
FOOD ADDITIVES AND CONTAMINANTS, 1991, VOL. 8, NO. 6, 713-722
Effects of harmane on growth and in vivo metabolism of aflatoxin B1 in male and female rats CATHERINE BILLAUD Laboratoire de Biochimie Industrielle et Agro-alimentaire, Conservatoire National des Arts et Métiers, 292, rue Saint-Martin, 75141 Paris Cedex 03, France
Downloaded by [University of Florida] at 07:39 08 November 2015
(Received 19 April 1991, revised 14 October 1991; accepted 11 November 1991) The role of harmane, a β-carboline formed during pyrolysis of tryptophan, on the metabolism of AFB1, growth and some parameters of the nutritional status was investigated in the rat. Male and female Wistar rats were fed a semi-synthetic diet containing AFB1 (2 ppm), harmane (250 ppm) or both compounds, for 33 days after weaning. Qualitative and quantitative differences in the urinary and faecal excretion of parental compound and metabolites were assessed by HPLC analysis. Harmane did not modify appreciably the growth and the other nutritional parameters studied. Similar excretion patterns of AFB1 metabolites were observed in males and females. Harmane caused a limited increase in the excretion of AFM1 in faeces but not in urine, without altering the growth process in rats of either sex. Keywords: Harmane, aflatoxin B1, metabolism, growth, rat Introduction
Aflatoxin Bi (AFBi) is a potent hepatotoxic and hepatocarcinogen for animals and possibly for man. Like many other toxins, AFBi requires metabolic activation to become oncogenic. Microsomal cytochrome(s) P450-linked monooxygenases are involved in the formation of highly reactive intermediates such as DNA-reactive epoxides (Essigman et al., 1982). These systems, in conjunction with cytosolic enzymes convert AFBi into various metabolites such as AFMi (hydroxylated form), AFPi (demethylated form) and aflatoxicol (Ro, reduced form). Some of these products may also be conjugated with glucuronic acid, sulphate or glutathione and excreted into bile, urine and faeces (Raj and Lotlikar, 1984). Both natural and synthetic compounds present in animal and human diets have been shown to reduce AFBi-induced carcinogenesis (Godlewski et al. 1985, Monroe et al. 1986, Wattenberg et al. 1986, Kensler et al. 1987, Bhattacharya and Firozi 1988, Goeger et al. 1988, Ramsdell and Eaton 1988, Fong and Bailey 1990). They could favour the formation of less carcinogenic metabolites such as AFMi and/or other readily extractable hydrophilic metabolites (Fukayama and Hsieh 1985). Contrastingly, numerous chemicals present in minute amounts in food are generated by food processing and may act as initiators of carcinogenesis. In this view, the cooking of protein-rich food and more particularly the pyrolysis of proteins and indole derivatives generate heterocyclic amines exhibiting mutagenic activity higher than AFBi and a carcinogenic potency, when given orally to rodents (Matsukura et al. 1981, Takayama et al. 1984, Sugimura 1988). 0265-203X/91 $3.00 © 1991 Taylor & Francis Ltd.
714
Catherine Billaud
o H H
R
1
R= H
X= H
NORHARMANE
2
R = CH 3
X= H
HARMANE
Downloaded by [University of Florida] at 07:39 08 November 2015
Figure 1. Structure of two 0-carbolines, harmane and norharmane.
The (3-carbolines harmane and norharmane devoid of exocyclic amino groups (figure 1), though not mutagenic per se, are known to enhance the genotoxicity of various chemicals (Nagao et al. 1977, Gupta et al. 1989). These compounds are found in various beverages and foodstuffs at levels ranging from 0-1 to 1000 ng/g (Adachi et al. 1991) and represent 8-10% by weight of the pyrolysis product of DL-tryptophan. In a previous experiment (unpublished results) we noticed that tryptophan pyrolysate when administered to rats at the dose of 2 g per kg of diet (i.e. 200 ppm approx. of harmane + norharmane), partly inhibited the hepatocarcinogenesis induced by AFBi. Moreover, in rats fed the tryptophan pyrolysate-enriched diet, we observed a decrease in the urinary excretion of AFMi, one of the major metabolites of AFBi. These effects were more pronounced in males than in females. In the present work, the effects of harmane on growth of male and female rats fed an AFBi-containing diet were studied. An attempt was also made to determine the identity and excretion level of some AFBi metabolites. Materials and methods
Animals Weaning Wistar CF rats of both sexes averaging 46 g body weight were purchased from R. Janvier breeding laboratory (Le Genest St Isle, France) and were divided into groups of 12 animals per treatment and per sex. Series of three or four rats were kept in stainless steel cages, males separated from females and fed ad libitum one of the four experimental diets. Chemicals and special diets The semi-synthetic basal diet used was prepared weekly in our laboratory; its composition is listed in table 1. In the experimental diets, a concentrated ethanolic solution of harmane (H) and/or aflatoxin Bi (AFBi) was thoroughly blended with the balanced mixture to provide a final concentration of 2 mg aflatoxin Bi and 250 mg harmane per kg of diet. The same amount of ethanol was incorporated to the control diet. Aflatoxin Bi (crystalline form) was purchased from Makor Chemicals (Jerusalem, Israel); harmane (l-methyl-9H-pyrido[3,4-b]indole) free base (crystalline form) came from Sigma Chemicals Company (St Louis, MO). Experimental design During the study, animals were divided into eight experimental groups as
Effects of harmane on growth and AFBi metabolism
715
Table 1. Composition of basal diet in control and experimental groups (identical for male and female rats) (g/kg).
Downloaded by [University of Florida] at 07:39 08 November 2015
Lactic casein Soyamin 90 Cellulose Cornstarch Vitamin mixture 8 Mineral mixture b Lard Edible soybean oil (ml) Methionine Choline 40 per 100 (ml) Sucrose q.s.
20 130 20 200 10 40 100 60 1 2-5 1000
a Vitamin mixture contained per kg: Vitamin A (retinol palmitate IU, 250000/g) 1-2 g; Vitamin D 2 or D 3 (IU 500000/g), 0-3 g; Vitamin E (DL-a-tocopherol succinic acid) (IU 1200/g), 3-3 g; Vitamin K3 (menadione), 300 mg; Vitamin Bi (thiamine HC1), 150 mg; Vitamin B 2 (riboflavin), 250 mg; vitamin PP or B3 (niacin) 3000 mg; vitamin B5 (calcium pantothenate) 500 mg; vitamin B6 (pyridoxine HC1) 150 mg; vitamin B7 (inositol), 10000 mg; vitamin H or Bg (D-biotin) 16 mg; vitamin Bc or B 9 (folic acid), 50 mg; vitamin B12 (cyanocobalamin), 50 mg; paraminobenzoic acid, 5000 mg; cornstarch q.s. 1000 g b L . Gueguen salt mixture (INRA, La Miniere: CM 5420).
follows: —two groups C (control): male or female rats given the plain semi-synthetic diet without chemical added throughout the experiment; —two groups H (harmane): male or female rats given the diet with harmane at the level of 250 ppm; —two groups AFBi (aflatoxin Bi): male or female rats given the diet with AFBt at the level of 2 ppm; —two groups AFBi + H (aflatoxin B t + harmane): male or female rats given the diet with AFBi plus harmane at the above mentioned concentrations. After a period of 28 days, male and female rats were randomly allocated to individual metabolism cages for 5 days. We used seven rats from C and H group and 12 from AFBi and AFBi + H. Food containers were weighed daily in order to evaluate the uptake of food and contaminants. Their body weight was recorded at the beginning and at the end of the metabolism period. Individual faecal and urine excretions were collected daily, weight and volumes recorded. When appropriated, gross observations of appearance and behaviour were noticed. Sampling of urine and faeces Urine samples from 5 days were collected free of faecal contamination into graduated vessels containing a few ml of toluene in order to protect chemical compounds from oxidation, thereafter specimens were each filtered through glass wool, then transferred to flasks and adjusted to a final 100 ml with distilled water.
716
Catherine Billaud
Downloaded by [University of Florida] at 07:39 08 November 2015
Pooled faeces from 5 days were dried in a lyophilizator and finely ground with aid of a mini ballgrinder. Both samples were kept frozen until determination of aflatoxins levels. Material Liquid chromatography apparatus. Reversed-phase chromatography was performed using Constametric IIG HPLC system equipped with Constametric I and II pumps (LDC Milton Roy), a model UHP - 7 K Valco injector (25 /il sample loop) and a gradient master solvent programmer (LDC). The aflatoxins were detected with a model 1311 Fluoromonitor III, fluorometric detector (LDC) equipped with an excitation filter (340-380 nm transmission), and emission filter (>417nm transmission) and a 30 /tl flow cell. Peak areas were calculated with a LDC 304 - 50 computing integrator. Separation of the aflatoxins was achieved on a 25 cmx4-6 mm i.d., 5 urn, C18 column (Vydac 201 TP 54, Hesperia, California), operating at 1-1 ml/min (nominal pressure of =2000 psi). Reagents. Reagent grade acetone, chloroform, dichloromethane, «-hexane, ethanol, benzene, diethylether and anhydrous sodium sulphate, HPLC grade acetonitrile were from Prolabo (Paris, France). For HPLC elution system solvent, analytical reagent methanol (Prolabo, France) was redistilled in our laboratory. HPLC elution solvent consisted of a water/methanol/acetonitrile mixture (70:15:15). The mixture was degassed by sonication just prior to analysis. Standard solutions. Aflatoxins were obtained from Makor Chemicals Ltd (Jerusalem, Israel), AFMi and AFBi from Aspergillus Jlavus, AFPi chemically derived from Aspergillus Jlavus and aflatoxicol I ( = Ro), natural isomer. Stock solutions of each aflatoxin were prepared in benzene/acetonitrile (9 + 1), and kept at -20°C in the dark. Mixed aflatoxin working solutions were diluted with methanol to provide appropriate concentrations. Extraction procedure Aliquots of urine (10 ml) were extracted twice, each time with 50 ml volumes of chloroform/acetone (1:1) and dried over 30 g anhydrous sodium sulphate. After evaporation of organic solvents using a rotavapor (Bu'chi) at a temperature
