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Amelioration of cytotoxic effects of aflatoxin by vitamin A: an in vitro study on erythrocytes
Toxicology in Vitro 15 (2001) 39±42
www.elsevier.com/locate/toxinvit
Brief communication
Amelioration of cytotoxic eects of a¯atoxin by vitamin A: an in vitro study on erythrocytes R.J. Verma a,*, R.S. Shukla b, D.N. Mehta b b
a Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad 380 009, India Silicates and Catalysis Division, Central Salt and Marine Chemicals Research Institute, Bhavnagar 364 002, India
Accepted 4 October 2000
Abstract We have evaluated the ameliorative role of vitamin A on a¯atoxin-induced cytotoxicity in vitro. A¯atoxin (1.95 mm)-induced hemolysis was found to be signi®cantly reduced on addition of vitamin A (125±1250 IU/ml) in incubation medium. The decrease in hemolysis was almost dose dependent. The kinetics of reduction of AFB1 to B2 and AFG1 to G2 by vitamin A has been investigated in dilute aqueous solution at 37 C. The rate of the reduction was found to be ®rst order with respect to the concentration of vitamin A and a¯atoxin concentration. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: A¯atoxin; Vitamin A; Cytotoxicity; Amelioration
A¯atoxins are food-borne, secondary toxic fungal metabolites produced by Aspergillus ¯avus and A. parasiticus. A¯atoxin B1, and to a lesser extent AFG1, are responsible for the biological potency of a¯atoxincontaminated meals and crude fractions derived from toxigenic A. ¯avus cultures. A¯atoxins B2 and AFG2 are essentially biologically inactive unless these agents are ®rst metabolically oxidized to AFB1 and AFG1 (Busby and Wogan, 1984; Pohland, 1993; Groopman et al., 1996). A¯atoxin B1 is oxidized by microsomal mixed-function oxidase (cytochrome P450) to several products. It is generally believed that formation of AFB1-8,9-epoxide, an active metabolite and its subsequent covalent bindings to DNA, RNA and proteins plays a critical role in a¯atoxin toxicities (Choy, 1993; Eaton and Gallagher, 1994). In addition, the modulating eects of vitamin A on the toxicity of a¯atoxin either by direct interaction or by in¯uencing the metabolism of a¯atoxin are well known (Decoudu et al., 1992; Sinha and Dharmshila, 1994). Our earlier studies (Verma and Raval, 1991) revealed a concentration-dependent increase in the rate of haemolysis indicating AFB1-induced cytotoxicity which could be due to lipid peroxidation of plasma membrane, Abbreviation: RBC, red blood cell * Corresponding author. E-mail address: [email protected] (R.J. Verma).
permeability alterations and cell lysis. Recent studies have shown that a¯atoxin causes lipid peroxidation and oxidative damage in liver and other vital organs which may involve hydroxyl radicals as the initiating species (Shen et al., 1994, 1995; Verma and Nair, 1999). Many investigators (Decoudu et al., 1993; Yu et al., 1994; Odin, 1997) have shown that vitamin A (retinol) can scavenge reactive oxy-radicals through their binding to polyene chain atoms or by way of free radical proton abstraction from the a-carbon to polyene chain atoms. However, the ecacy of vitamin A, an important fatsoluble bio-antioxidant on a¯atoxin-induced cytotoxicities, especially on erythrocytes, which lack microsomal mixed-function oxidase required for bioactivation of AFB1, remain unknown. The aim of the present investigation was to evaluate the ameliorative eect of vitamin A (Aquasol A) on a¯atoxin-induced cytotoxicities. A toxigenic strain of A. parasiticus (NRRL-3240), obtained from the Indian Agricultural Research Institute, New Delhi, was grown on sucrose-magnesium sulfate-potassium nitrate-yeast extract (SMKY) liquid medium at 282 C for 10 days (Diener and Davis, 1966). Pooled culture ®ltrates were extracted with analytical grade chloroform (1:2, v/v) and passed through anhydrous sodium sulfate bed. The chloroform extract was evaporated to dryness and stored. All the chemicals used were of A.R. grade and A.R. grade vitamin A (Aquasol A) was used without further puri®cation. All
0887-2333/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0887-2333(00)00058-8
40
R.J. Verma et al. / Toxicology in Vitro 15 (2001) 39±42
the reactions were run in freshly prepared double-distilled water. The residue was dissolved in fresh chloroform and used for a¯atoxin quanti®cation. The solution (100 ml) along with pure a¯atoxin standards (a gift from the International Agency for Research on Cancer, Lyon, France) were spotted on silica gel G coated activated TLC plates. Thereafter, the plates were developed in a solvent consisting of toluene:iso-amyl alcohol: methanol (90:32:2, by vol.) (Reddy et al., 1970). The plates were air-dried and observed under long-wave UV light (360 nm) for a¯atoxins (B1, B2, G1 and G2). The a¯atoxins were chemically con®rmed by spraying tri¯uoroacetic acid or 25% sulfuric acid. Each spot was eluted separately and dissolved in chilled methanol. A¯atoxins were quanti®ed using Shimadzu 160 UV-Vis spectrophotometer at 360 nm (Nabney and Nesbitt, 1965). The aqueous solution of a¯atoxin contained a mixture of a¯atoxin B1, B2, G1 and G2 in the ratio of 30:20:30:20. Five ml of blood collected in EDTA bulb from the ear pinna of adult New Zealand strain rabbits (Oryctolagus cuniculus) was used for the preparation of red blood cell (RBC) suspension in normal saline (0.9% NaCl), giving a cell density of 2104 cells/ml (Verma and Raval, 1991). To test the ecacy of vitamin A in prevention of a¯atoxin-induced hemolysis, 1 ml of RBC suspensions were added with (a) vitamin A 125 IU/ml, (b) 1.95 mm a¯atoxin (Verma and Raval, 1991), or (c) 1.95 mm a¯atoxin and 125±1250 IU/ml vitamin A. The volume of each tube was made up to 2 ml with additional saline. The required concentrations of vitamin A and a¯atoxins were prepared in normal saline. Tubes were mixed gently and incubated in a shaking water-bath at 37 C for 16 h. Thereafter, the tubes were centrifuged at 300 g for 10 min and the colour density of the supernatant was measured spectrophotometrically at 540 nm. The percent hemolysis was calculated by the formula: Percent hemolysis Absorbance of the individual tubes 100 Absorbance with 100% hemolysis To achieve 100% hemolysis, 1.0 ml distilled water was added to 1 ml RBC suspension (Verma and Raval, 1991). Morphologically, the RBC appear as biconcave discs or indented spheres in control tubes. The cells remain settled in the bottom of the tube with clear supernatant. The addition of 1.95 mm a¯atoxin to RBC suspension caused pronounced swelling and cell lysis. The cells pellet in the bottom of the tube reduced with a reddishcoloured supernatant indicating hemolysis. The rise in percent hemolysis was signi®cantly dierent as compared with the control (Table 1). Concurrent addition of vitamin A (125±1250 IU/ml) and 1.95 mm a¯atoxin in RBC suspension signi®cantly
reduced a¯atoxin-induced hemolysis. Except at 500 and 625 IU/ml concentration, a dose-dependent response was observed. The maximum (55%) reduction was noted at 1000 IU/ml vitamin A; a further rise in concentration comparatively decreased percent reduction (Table 1). In another set of experiments, an aqueous solution of 3.89 mm a¯atoxin was mixed thoroughly with 10,000 IU/ ml vitamin A and incubated at 37 C. An excess of vitamin A was used in kinetic runs to ensure pseudo-®rst order kinetic conditions. At regular intervals, 100 ml of reaction mixture (in triplicate) was spotted on activated TLC plates for quantifying time-dependent changes in concentration of a¯atoxins. Changes in the concentration of AFB1 to B2 (Fig. 1) and AFG1 to G2 (Fig. 2) revealed a signi®cant decrease in AFB1 and AFG1, followed by an increase in concentration of AFB2 and AFG2. Initial rates of decrease in AFB1 (9.510 8 m/h) and AFG1 (1010 8 m/h) were much higher than at the later phase. The rate of increase in concentration of AFB2 (2.510 8 m/h) and AFG2 (2.1610 8 m/h) was almost uniform. The present experiment clearly indicates that vitamin A interacts with a¯atoxin B1 and AFG1 and converts it into inactive AFB2 and AFG2. In addition, vitamin A can scavenge reactive oxy-radicals (Decoudu et al., 1993; Yu et al., 1994; Odin, 1997) and thereby reduce lipid peroxidation and haemolysis. Vitamin C acts similarly to vitamin A (Verma et al., 1999). In conclusion, vitamin A is observed as an eective reductant for percentage reduction of hemolysis and cytotoxicity of AFB1 and AFG1. The reaction of dilute solution of vitamin A and a¯atoxin leads the reduction of AFB1 to AFB2 and AFG1 to AFG2, which could be responsible for reduction in a¯atoxin-induced cytotoxicities.
Table 1 Reduction of a¯atoxin-induced hemolysis by vitamin A in vitroa Treatments A¯atoxin (mm)
Vitamin A (IU/ml)
Percent hemolysis
Reduction (%)
0.0 0.0 1.95 1.95 1.95 1.95 1.95 1.95 1.95 1.95 1.95 1.95 1.95
0.0 125 0.0 125 250 375 500 625 750 875 1000 1125 1250
1.851.00 1.80 0.89 62.88 1.06b* 49.52 0.64c* 41.11 0.74c* 35.53 0.68c* 42.59 0.70c* 40.37 0.80c* 35.19 0.59c* 30.46 0.94c* 28.90 1.72c* 38.27 1.20c* 42.66 1.35c*
± ± ± 21.24 34.62 43.49 32.26 35.79 44.03 51.55 54.03 39.13 32.15
a
MeanS.E.M.; n=5). As compared with control. c As compared with a¯atoxin treated. *P<0.001. b
R.J. Verma et al. / Toxicology in Vitro 15 (2001) 39±42
41
Fig. 1. Time-dependent changes in concentration of a¯atoxin B1 and B2 and G1 and G2.
Fig. 2. G1 and G2 during interaction with vitamin A. Aqueous solution of 3.89 mm a¯atoxin containing AFB1 B2, G1 and G2 in the concentrations of 1.2, 0.8, 1.14 and 0.75 mm, respectively, was exposed to 10,000 IU/ml vitamin A.
References Busby, W.F., Wogan, G.N., 1984. A¯atoxins. In: Searle, S.E. (Ed.), Chemical Carcinogens. ACS Monograph 182, American Chemical Society, Washington, DC, pp. 945±1136. Choy, W.N., 1993. A review of the dose response induction of DNA adducts by a¯atoxin B1 and its implications to quantitative cancer risk assessment. Mutation Research 296, 181±198. Decoudu, S., Cassand, P., Colin, V., Koch, B., Narbonne, J.F., 1993. Eect of vitamin A dietary intake on DNA damage induced in vitro by AFB1. Special Publ. Royal Society of Chemistry 123, 130±132. Decoudu, S., Cassand, P., Daubeze, M., Frayssinet, C., Narbonne, J.F., 1992. Eect of vitamin A dietary intake on in vitro and in vivo activation of a¯atoxin B1. Mutation Research 269, 269±278.
Diener, U.L., Davis, N.D., 1966. A¯atoxin production by isolates of Aspergillus ¯avus. Phytopathology 56, 1390±1393. Eaton, D.L., Gallagher, E.P., 1994. Mechanisms of a¯atoxin carcinogenesis. Annual Review of Pharmacology and Toxicology 34, 135±172. Groopman, J.D., Wang, J.S., Scholl, P., 1996. Molecular biomarkers for a¯atoxins: From adducts to gene mutations to human liver cancer. Canadian Journal of Physiology and Pharmacology 74, 203±209. Nabney, J., Nesbitt, B.F., 1965. A spectrophotometric method for determining the a¯atoxins. Analysis 90, 155±160. Odin, A.P., 1997. Vitamins as antimutagens: Advantages and some possible mechanisms of antimutagenic action. Mutation Research 386, 39±67. Pohland, A.E., 1993. Mycotoxins in review. Food Additives and Contaminants 10, 17±28.
42
R.J. Verma et al. / Toxicology in Vitro 15 (2001) 39±42
Reddy, T.V., Vishwanathan, L., Venkitasubramanian, T.A., 1970. Thin layer chromatography of a¯atoxins. Analytical Biochemistry 38, 568±571. Shen, H.M., Shi, C.Y., Lee, H.P., Ong, C.N., 1994. A¯atoxin B1induced lipid peroxidation in rat liver. Toxicology and Applied Pharmacology 127, 145±150. Shen, H.M., Ong, C.H., Lee, B.L., Shi, C.Y., 1995. A¯atoxin B1induced 8-hydroxydeoxyguanosine formation in rat hepatic DNA. Carcinogenesis 16, 419±422. Sinha, S.P., Dharamshila, K., 1994. Vitamin A ameliorates the genotoxicity in mice of a¯atoxin B1-containing Aspergillus ¯avus infested food. Cytobios 79, 85±95.
Verma, R.J., Nair, A., 1999. Vitamin E prevents a¯atoxin-induced lipid peroxidation in the liver and kidney. Medical Science Research 27, 223±226. Verma, R.J., Raval, P.J., 1991. Cytotoxicity of a¯atoxin on red blood corpuscles. Bulletin of Environmental Contamination and Toxicology 47, 428±433. Verma, R.J., Shukla, R.S., Mehta, D.N., 1999. Interaction of a¯atoxin with l-ascorbic acid: a kinetic and mechanistic approach. Natural Toxins 7, 25±29. Yu, M.W., Zhang, Y.J., Blaner, W.S., Sarilella, R.M., 1994. In¯uence of vitamin A, C, E and b-carotene on a¯atoxin B1 binding to DNA in wood chunk hepatocytes. Cancer 73, 596±604.
www.elsevier.com/locate/toxinvit
Brief communication
Amelioration of cytotoxic eects of a¯atoxin by vitamin A: an in vitro study on erythrocytes R.J. Verma a,*, R.S. Shukla b, D.N. Mehta b b
a Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad 380 009, India Silicates and Catalysis Division, Central Salt and Marine Chemicals Research Institute, Bhavnagar 364 002, India
Accepted 4 October 2000
Abstract We have evaluated the ameliorative role of vitamin A on a¯atoxin-induced cytotoxicity in vitro. A¯atoxin (1.95 mm)-induced hemolysis was found to be signi®cantly reduced on addition of vitamin A (125±1250 IU/ml) in incubation medium. The decrease in hemolysis was almost dose dependent. The kinetics of reduction of AFB1 to B2 and AFG1 to G2 by vitamin A has been investigated in dilute aqueous solution at 37 C. The rate of the reduction was found to be ®rst order with respect to the concentration of vitamin A and a¯atoxin concentration. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: A¯atoxin; Vitamin A; Cytotoxicity; Amelioration
A¯atoxins are food-borne, secondary toxic fungal metabolites produced by Aspergillus ¯avus and A. parasiticus. A¯atoxin B1, and to a lesser extent AFG1, are responsible for the biological potency of a¯atoxincontaminated meals and crude fractions derived from toxigenic A. ¯avus cultures. A¯atoxins B2 and AFG2 are essentially biologically inactive unless these agents are ®rst metabolically oxidized to AFB1 and AFG1 (Busby and Wogan, 1984; Pohland, 1993; Groopman et al., 1996). A¯atoxin B1 is oxidized by microsomal mixed-function oxidase (cytochrome P450) to several products. It is generally believed that formation of AFB1-8,9-epoxide, an active metabolite and its subsequent covalent bindings to DNA, RNA and proteins plays a critical role in a¯atoxin toxicities (Choy, 1993; Eaton and Gallagher, 1994). In addition, the modulating eects of vitamin A on the toxicity of a¯atoxin either by direct interaction or by in¯uencing the metabolism of a¯atoxin are well known (Decoudu et al., 1992; Sinha and Dharmshila, 1994). Our earlier studies (Verma and Raval, 1991) revealed a concentration-dependent increase in the rate of haemolysis indicating AFB1-induced cytotoxicity which could be due to lipid peroxidation of plasma membrane, Abbreviation: RBC, red blood cell * Corresponding author. E-mail address: [email protected] (R.J. Verma).
permeability alterations and cell lysis. Recent studies have shown that a¯atoxin causes lipid peroxidation and oxidative damage in liver and other vital organs which may involve hydroxyl radicals as the initiating species (Shen et al., 1994, 1995; Verma and Nair, 1999). Many investigators (Decoudu et al., 1993; Yu et al., 1994; Odin, 1997) have shown that vitamin A (retinol) can scavenge reactive oxy-radicals through their binding to polyene chain atoms or by way of free radical proton abstraction from the a-carbon to polyene chain atoms. However, the ecacy of vitamin A, an important fatsoluble bio-antioxidant on a¯atoxin-induced cytotoxicities, especially on erythrocytes, which lack microsomal mixed-function oxidase required for bioactivation of AFB1, remain unknown. The aim of the present investigation was to evaluate the ameliorative eect of vitamin A (Aquasol A) on a¯atoxin-induced cytotoxicities. A toxigenic strain of A. parasiticus (NRRL-3240), obtained from the Indian Agricultural Research Institute, New Delhi, was grown on sucrose-magnesium sulfate-potassium nitrate-yeast extract (SMKY) liquid medium at 282 C for 10 days (Diener and Davis, 1966). Pooled culture ®ltrates were extracted with analytical grade chloroform (1:2, v/v) and passed through anhydrous sodium sulfate bed. The chloroform extract was evaporated to dryness and stored. All the chemicals used were of A.R. grade and A.R. grade vitamin A (Aquasol A) was used without further puri®cation. All
0887-2333/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0887-2333(00)00058-8
40
R.J. Verma et al. / Toxicology in Vitro 15 (2001) 39±42
the reactions were run in freshly prepared double-distilled water. The residue was dissolved in fresh chloroform and used for a¯atoxin quanti®cation. The solution (100 ml) along with pure a¯atoxin standards (a gift from the International Agency for Research on Cancer, Lyon, France) were spotted on silica gel G coated activated TLC plates. Thereafter, the plates were developed in a solvent consisting of toluene:iso-amyl alcohol: methanol (90:32:2, by vol.) (Reddy et al., 1970). The plates were air-dried and observed under long-wave UV light (360 nm) for a¯atoxins (B1, B2, G1 and G2). The a¯atoxins were chemically con®rmed by spraying tri¯uoroacetic acid or 25% sulfuric acid. Each spot was eluted separately and dissolved in chilled methanol. A¯atoxins were quanti®ed using Shimadzu 160 UV-Vis spectrophotometer at 360 nm (Nabney and Nesbitt, 1965). The aqueous solution of a¯atoxin contained a mixture of a¯atoxin B1, B2, G1 and G2 in the ratio of 30:20:30:20. Five ml of blood collected in EDTA bulb from the ear pinna of adult New Zealand strain rabbits (Oryctolagus cuniculus) was used for the preparation of red blood cell (RBC) suspension in normal saline (0.9% NaCl), giving a cell density of 2104 cells/ml (Verma and Raval, 1991). To test the ecacy of vitamin A in prevention of a¯atoxin-induced hemolysis, 1 ml of RBC suspensions were added with (a) vitamin A 125 IU/ml, (b) 1.95 mm a¯atoxin (Verma and Raval, 1991), or (c) 1.95 mm a¯atoxin and 125±1250 IU/ml vitamin A. The volume of each tube was made up to 2 ml with additional saline. The required concentrations of vitamin A and a¯atoxins were prepared in normal saline. Tubes were mixed gently and incubated in a shaking water-bath at 37 C for 16 h. Thereafter, the tubes were centrifuged at 300 g for 10 min and the colour density of the supernatant was measured spectrophotometrically at 540 nm. The percent hemolysis was calculated by the formula: Percent hemolysis Absorbance of the individual tubes 100 Absorbance with 100% hemolysis To achieve 100% hemolysis, 1.0 ml distilled water was added to 1 ml RBC suspension (Verma and Raval, 1991). Morphologically, the RBC appear as biconcave discs or indented spheres in control tubes. The cells remain settled in the bottom of the tube with clear supernatant. The addition of 1.95 mm a¯atoxin to RBC suspension caused pronounced swelling and cell lysis. The cells pellet in the bottom of the tube reduced with a reddishcoloured supernatant indicating hemolysis. The rise in percent hemolysis was signi®cantly dierent as compared with the control (Table 1). Concurrent addition of vitamin A (125±1250 IU/ml) and 1.95 mm a¯atoxin in RBC suspension signi®cantly
reduced a¯atoxin-induced hemolysis. Except at 500 and 625 IU/ml concentration, a dose-dependent response was observed. The maximum (55%) reduction was noted at 1000 IU/ml vitamin A; a further rise in concentration comparatively decreased percent reduction (Table 1). In another set of experiments, an aqueous solution of 3.89 mm a¯atoxin was mixed thoroughly with 10,000 IU/ ml vitamin A and incubated at 37 C. An excess of vitamin A was used in kinetic runs to ensure pseudo-®rst order kinetic conditions. At regular intervals, 100 ml of reaction mixture (in triplicate) was spotted on activated TLC plates for quantifying time-dependent changes in concentration of a¯atoxins. Changes in the concentration of AFB1 to B2 (Fig. 1) and AFG1 to G2 (Fig. 2) revealed a signi®cant decrease in AFB1 and AFG1, followed by an increase in concentration of AFB2 and AFG2. Initial rates of decrease in AFB1 (9.510 8 m/h) and AFG1 (1010 8 m/h) were much higher than at the later phase. The rate of increase in concentration of AFB2 (2.510 8 m/h) and AFG2 (2.1610 8 m/h) was almost uniform. The present experiment clearly indicates that vitamin A interacts with a¯atoxin B1 and AFG1 and converts it into inactive AFB2 and AFG2. In addition, vitamin A can scavenge reactive oxy-radicals (Decoudu et al., 1993; Yu et al., 1994; Odin, 1997) and thereby reduce lipid peroxidation and haemolysis. Vitamin C acts similarly to vitamin A (Verma et al., 1999). In conclusion, vitamin A is observed as an eective reductant for percentage reduction of hemolysis and cytotoxicity of AFB1 and AFG1. The reaction of dilute solution of vitamin A and a¯atoxin leads the reduction of AFB1 to AFB2 and AFG1 to AFG2, which could be responsible for reduction in a¯atoxin-induced cytotoxicities.
Table 1 Reduction of a¯atoxin-induced hemolysis by vitamin A in vitroa Treatments A¯atoxin (mm)
Vitamin A (IU/ml)
Percent hemolysis
Reduction (%)
0.0 0.0 1.95 1.95 1.95 1.95 1.95 1.95 1.95 1.95 1.95 1.95 1.95
0.0 125 0.0 125 250 375 500 625 750 875 1000 1125 1250
1.851.00 1.80 0.89 62.88 1.06b* 49.52 0.64c* 41.11 0.74c* 35.53 0.68c* 42.59 0.70c* 40.37 0.80c* 35.19 0.59c* 30.46 0.94c* 28.90 1.72c* 38.27 1.20c* 42.66 1.35c*
± ± ± 21.24 34.62 43.49 32.26 35.79 44.03 51.55 54.03 39.13 32.15
a
MeanS.E.M.; n=5). As compared with control. c As compared with a¯atoxin treated. *P<0.001. b
R.J. Verma et al. / Toxicology in Vitro 15 (2001) 39±42
41
Fig. 1. Time-dependent changes in concentration of a¯atoxin B1 and B2 and G1 and G2.
Fig. 2. G1 and G2 during interaction with vitamin A. Aqueous solution of 3.89 mm a¯atoxin containing AFB1 B2, G1 and G2 in the concentrations of 1.2, 0.8, 1.14 and 0.75 mm, respectively, was exposed to 10,000 IU/ml vitamin A.
References Busby, W.F., Wogan, G.N., 1984. A¯atoxins. In: Searle, S.E. (Ed.), Chemical Carcinogens. ACS Monograph 182, American Chemical Society, Washington, DC, pp. 945±1136. Choy, W.N., 1993. A review of the dose response induction of DNA adducts by a¯atoxin B1 and its implications to quantitative cancer risk assessment. Mutation Research 296, 181±198. Decoudu, S., Cassand, P., Colin, V., Koch, B., Narbonne, J.F., 1993. Eect of vitamin A dietary intake on DNA damage induced in vitro by AFB1. Special Publ. Royal Society of Chemistry 123, 130±132. Decoudu, S., Cassand, P., Daubeze, M., Frayssinet, C., Narbonne, J.F., 1992. Eect of vitamin A dietary intake on in vitro and in vivo activation of a¯atoxin B1. Mutation Research 269, 269±278.
Diener, U.L., Davis, N.D., 1966. A¯atoxin production by isolates of Aspergillus ¯avus. Phytopathology 56, 1390±1393. Eaton, D.L., Gallagher, E.P., 1994. Mechanisms of a¯atoxin carcinogenesis. Annual Review of Pharmacology and Toxicology 34, 135±172. Groopman, J.D., Wang, J.S., Scholl, P., 1996. Molecular biomarkers for a¯atoxins: From adducts to gene mutations to human liver cancer. Canadian Journal of Physiology and Pharmacology 74, 203±209. Nabney, J., Nesbitt, B.F., 1965. A spectrophotometric method for determining the a¯atoxins. Analysis 90, 155±160. Odin, A.P., 1997. Vitamins as antimutagens: Advantages and some possible mechanisms of antimutagenic action. Mutation Research 386, 39±67. Pohland, A.E., 1993. Mycotoxins in review. Food Additives and Contaminants 10, 17±28.
42
R.J. Verma et al. / Toxicology in Vitro 15 (2001) 39±42
Reddy, T.V., Vishwanathan, L., Venkitasubramanian, T.A., 1970. Thin layer chromatography of a¯atoxins. Analytical Biochemistry 38, 568±571. Shen, H.M., Shi, C.Y., Lee, H.P., Ong, C.N., 1994. A¯atoxin B1induced lipid peroxidation in rat liver. Toxicology and Applied Pharmacology 127, 145±150. Shen, H.M., Ong, C.H., Lee, B.L., Shi, C.Y., 1995. A¯atoxin B1induced 8-hydroxydeoxyguanosine formation in rat hepatic DNA. Carcinogenesis 16, 419±422. Sinha, S.P., Dharamshila, K., 1994. Vitamin A ameliorates the genotoxicity in mice of a¯atoxin B1-containing Aspergillus ¯avus infested food. Cytobios 79, 85±95.
Verma, R.J., Nair, A., 1999. Vitamin E prevents a¯atoxin-induced lipid peroxidation in the liver and kidney. Medical Science Research 27, 223±226. Verma, R.J., Raval, P.J., 1991. Cytotoxicity of a¯atoxin on red blood corpuscles. Bulletin of Environmental Contamination and Toxicology 47, 428±433. Verma, R.J., Shukla, R.S., Mehta, D.N., 1999. Interaction of a¯atoxin with l-ascorbic acid: a kinetic and mechanistic approach. Natural Toxins 7, 25±29. Yu, M.W., Zhang, Y.J., Blaner, W.S., Sarilella, R.M., 1994. In¯uence of vitamin A, C, E and b-carotene on a¯atoxin B1 binding to DNA in wood chunk hepatocytes. Cancer 73, 596±604.