This article was downloaded by: [New York University] On: 02 June 2015, At: 11:40 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Some interactions of light, riboflavin, and aflatoxin B1 in vivo and in vitro a
a
Patricia I. Joseph‐Bravo , Margaret Findley & Paul M. Newberne
b
a
Department of Nutrition and Food Science , Massachusetts Institute of Technology , Cambridge, Massachusetts b
Department of Nutrition and Food Science , Massachusetts Institute of Technology , Cambridge, Massachusetts, 02139 Published online: 20 Oct 2009.
To cite this article: Patricia I. Joseph‐Bravo , Margaret Findley & Paul M. Newberne (1976) Some interactions of light, riboflavin, and aflatoxin B1 in vivo and in vitro , Journal of Toxicology and Environmental Health: Current Issues, 1:3, 353-376, DOI: 10.1080/15287397609529336 To link to this article: http://dx.doi.org/10.1080/15287397609529336
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
SOME INTERACTIONS OF LIGHT, RIBOFLAVIN, AND AFLATOXIN B1 IN VIVO AND IN VITRO Patricia I. Joseph-Bravo, Margaret Findley, Paul M. Newberne
Downloaded by [New York University] at 11:40 02 June 2015
Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts In previous studies, artificial sunlight and riboflavin synerglstically increased acute aflatoxin toxIcity in rats. Three new experiments were designed to provide information on the interaction of riboflavin, aflatoxin, and light. In a study of carcInogenesis, rats received low levels of aflatoxin 5 days/wk for 3 wk; 30 min after each dosing, half of them were Irradiated for 2 hr. In some, levels of glucose-6-phosphatase and acid phosphatase were determined 5 days after completion of treatment. Remaining rats were killed at 30 or 53 wk. All underwent complete necropsies and histopathologic examination. In the second experiment, rats were dosed with riboflavin and divided into four groups: no further treatment; aflatoxin (LD50); irradiation (1-2 hr); or aflatoxin plus irradiation. Blood riboflavin levels were determined at intervals following these treatments. In the third experiment, the chemical reactions of irradiated aflatoxin and/or riboflavin were studied by uv spectroscopy and TLC. The 53-wk study showed clearly that light decreased the incidence of aflatoxin-induced cancer. The other results may provide an explanation. Aflatoxin caused blood riboflavin levels to decrease-an effect enhanced by irradiation, suggesting that photosensitized riboflavin and aflatoxin form a complex. This interpretation gains support from studies in vitro that showed that riboflavin quenched aflatoxin photodegradation, perhaps by complexing with aflatoxin. Thus, low, carcinogenic doses of aflatoxin may complex with endogenous, photosensitized riboflavin, inhibiting its degradation into carcinogenic metabolites.
INTRODUCTION In 1964, Epstein et al. described a correlation between the photodynamic and carcinogenic activities of polycyclic compounds. Aflatoxin is a photosensitive compound; light, either directly or through the agency of such light-sensitive substances as carotenes or riboflavin, is known to influence the effects of aflatoxin in vivo (Neely et al., 1970; Smith and Neely, 1972). Previous studies (Newberne et al., 1974) have shown that artificial sunlight increases the toxicity of aflatoxin Bj in rats and that light plus excess orally administered riboflavin synergistically increase toxicity. Although aflatoxin occurs in relatively high concentrations in tropical and subtropical countries, exposed populations do not all respond the same way. Other environmental factors, such as diet (e.g., dietary These studies were supported by USPHS grants ES 00616 and ES 00497 and by a grant from the Duro-Test Corporation. Requests for reprints should be sent to Paul M. Newberne, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. 353 Journal of Toxicology and Environmental Health, 1:353-376, 1976 Copyright © 1976 by Hemisphere Publishing Corporation
Downloaded by [New York University] at 11:40 02 June 2015
354
P. I. JOSEPH-BRAVO ET AL.
riboflavin levels) and exposure to sunlight, may be important in determining biologic response. We have conducted a group of experiments designed to develop new information about the interactions among aflatoxin, riboflavin, and light in the living organism. The first experiment was a study of the effects of light on aflatoxin carcinogenicity in rats. Since some liver enzymes are believed to take part in the catabolism of aflatoxin and, perhaps, the formation of carcinogenic metabolites (Allison, 1966; Danilov et al., 1970), activities of glucose-6phosphatase and acid phosphatase were examined in some rats. In the second experiment, the effects of aflatoxin and light on blood levels of riboflavin-administered rats were measured. Whether light influences the metabolism of riboflavin was questioned by Maslenikova (1962), who studied the effect of solar radiation on the levels of riboflavin in blood, organs, and urine of rats receiving 20 jug vitamin/day; urinary excretion of riboflavin was increased and the hemoglobin concentration was decreased. Ultraviolet irradiation also caused a decrease in the levels of riboflavin in the blood of animals dosed with it. Similarly, X-rays increased riboflavin excretion by rats. Correlations between levels of riboflavin and the carcinogenicity of some compounds have been reported (Rivlin, 1973). Studies have indicated that excess riboflavin protects against, and deficiency potentiates, the hepatic carcinogenicity of 4-dimethylazobenzene (DAB) (Kensler et al., 1941; Lambooy, 1970; Mulay and O'Gara, 1968). On the other hand, Rubenchik (1963) has reported that DAB intensifies the excretion of riboflavin, an effect apparently due to an increase in toxicity of nucleotide phosphatase (Yang and Sung, 1966). Riboflavin deficiency affects the microsomal oxidative and reductive pathways (Shargel and Mazel, 1973), but riboflavin treatment of well-nourished rats produced no significant alterations in microsomal enzyme activities (Gillette et al., 1972), perhaps because much of the. riboflavin injected ¡ntraperitoneally in rats is destroyed or rapidly eliminated (Bessey et al., 1958; Christensen, 1969). The formation of a complex between riboflavin and other compounds has been observed by Radda (1966). Tung and Lin (1964) have observed a quenching effect of two azo dyes, DAB and 4-aminobenzene, on riboflavin fluorescence and have proposed the formation of a complex between these drugs and riboflavin in aqueous solution. This suggested that a direct complexing may block the action of the toxin at tissue receptor sites and thus modify biologic response to the toxin. In a third experiment, the chemical interactions of aflatoxin, riboflavin, and light were studied in vitro. Having long suspected that the metabolites responsible for aflatoxin's toxicity and carcinogenicity are different, we reasoned that analysis of reaction products might indicate whether metabolites vary under different environmental conditions, such as excess riboflavin and exposure to light.
Downloaded by [New York University] at 11:40 02 June 2015
LIGHT, RIBOFLAVIN, AND AFLATOXIN B,
355
Using ultraviolet spectroscopy and thin-layer chromatography (TLC), we examined irradiated and unirradiated samples of aflatoxin, riboflavin, and aflatoxin-riboflavin mixtures. Methanol was one of the solvents used. Because we wished to produce conditions similar to those found in vivo, we also studied the reaction in the presence of water. Since aflatoxin is not sufficiently soluble in water alone, a mixture of dimethylsulfoxide (DMSO) and water was used. Song et al. (1972) have reported that potassium iodide at concentrations of 10~ 4 M quenches the photoreduction of riboflavin when riboflavin is in the excited triplet state. This results from the formation of a strong complex between the alloxazine moiety of riboflavin and iodide in a proportion of 1:1. To determine whether this was the case in our system, we evaluated the influence of potassium iodide on the photoreduction of riboflavin in the riboflavin-aflatoxin mixture. The influence of a known inhibitor of riboflavin photoreduction, ascorbic acid (Reusser, 1970), was also examined. As a final check on the importance of the solvent used, aflatoxin and the aflatoxin-riboflavin mixture were dissolved in chloroform, irradiated, and subjected to TLC. METHODS Studies of Carcinogenesis Sixty male weanling rats of the Charles River CD strain (SpragueDawley, Charles River Breeding Laboratories) were housed in stainless steel cages and supplied with tap water ad libitum. Their weights were recorded periodically. All animal rooms were kept at 72°F and under controlled lighting (12-hr cycle). The complete, semisynthetic diet used throughout the experiment (Rogers and Newberne, 1971) was fed for 1 wk prior to initiation of aflatoxin treatment. Each rat was dosed intragastrically with 25 /ig of aflatoxin B r (Makor Chemicals, Jerusalem, Israel) in 0.2 ml DMSO (Fischer Scientific), 5 days/wk for 3 wk, to receive a total carcinogenic dose of 375 jug. Control animals received 0.2 ml DMSO. Thirty minutes after each aflatoxin administration, half of the rats from each group were irradiated for 2 hr with a long-arc xenon lamp (Vita-Lite, Duro-Test Corp.) with a filter combination giving a spectral distribution in the ultraviolet and visible ranges closely approximating natural light (Newberne et al., 1974). To attenuate the strong infrared resonance line of xenon, a water cell of plastic material (approximately 5 cm) was placed 50 cm below the lamp. Five days after the last aflatoxin dose was given, five rats from each group were decapitated, complete necropsies performed, and organs (liver, kidney, spleen, stomach, testes, heart, lung, and thymus) removed for histopathologic evaluation. Samples of liver were prepared in one of three
Downloaded by [New York University] at 11:40 02 June 2015
356
P. I. JOSEPH-BRAVO ET AL.
ways: fixed in formalin; frozen in liquid nitrogen for biochemical analysis of glucose-6-phosphatase activity and for histochemical determination of glucose-6-phosphatase and acid phosphatase; or used for immediate determination of acid phosphatase activity. Tissue concentration of acid phosphatase was determined by the method described in Sigma Technical Bulletin No. 104 (1968). For histochemical assay, the Gomori method was used (Barka and Anderson, 1965). The activity of glucose-6-phosphatase was determined according to the method of Harper (1965). For histochemical assay, the method of Wachstein and Merzel (1956) was employed. All reagents were purchased from the Sigma Chemical Company. Thirty weeks after the last aflatoxin dose, 10 rats from each group were decapitated, necropsies were performed, and organs and tissues removed for histopathologic examination. The remaining rats were sacrificed and studied 53 wk after aflatoxin treatment. Any rat that died during the 53-wk period was autopsied and its tissues examined histologically. Effect of Light and Aflatoxin on Riboflavin Levels The housing conditions, diet, and strain of rat used were the same as described above. On the day of the experiment, riboflavin-water solutions were prepared under dim light; the vitamin was dissolved in tubes covered with aluminum foil. The rats were dosed intragastrically with 1 or 10 mg riboflavin (General Biochemicals) per kilogram body weight, and riboflavin levels were measured in blood at appropriate intervals. The effect of aflatoxin on riboflavin levels was studied in 10 rats that received an L D 5 0 of aflatoxin (5 mg/kg body weight; Makor Chemicals, Jerusalem, Israel) in 0.2 ml DMSO, 45 min after they had received the riboflavin. Half the rats were irradiated for 1-2 hr. Rats were anesthetized with ether and bled by heart puncture. Riboflavin levels were determined in whole blood with an Aminco fluoromicrophotometer; the method of Clarke (1969), modified to use 6 ml of blood instead of 12 ml, was employed. Chemical Reactions in Vitro Methanol as solvent. Aflatoxin B! (Makor Chemicals) was dissolved in methanol (Allied Chemical Corp.), to a concentration of 1.78 X 10~ 4 M, in scintillation vials covered with aluminum foil. This concentration was chosen according to the extinction coefficient (approximately 2,000) to yield an absorbance value greater than 1. Because we wished to observe a 1:1 interaction, riboflavin (General Biochemicals) was dissolved under the same conditions to give a final concentration of 1.78 X 10~ 4 M. The solutions were placed under the irradiation source (as described above) in
Downloaded by [New York University] at 11:40 02 June 2015
LIGHT, RIBOFLAVIN, AND AFLATOXIN B,
357
uncovered vials for periods ranging from 15 min to 5 hr. When aflatoxin was irradiated in the presence of riboflavin, care was taken to have the same final concentration of 1.78 X 10~ 4 M. At the end of the irradiation, the vials were covered with aluminum foil and caps, and the spectrum was recorded immediately in a Gilford spectrophotometer 240, or spotted on TLC plates. DMSO-water as so/vent. Because DMSO is present in the blood of rats 2 hr after its administration as a solvent for aflatoxin (Newberne et al., 1974), the lowest proportion of DMSO to water possible without precipitation was used. Aflatoxin was dissolved first in the DMSO (Fischer Scientific), to which water was gradually added to give a final concentration of 10% (v/v). In the riboflavin solutions, DMSO was added in the same proportions. When the mixture was irradiated in the presence of potassium iodide (at a concentration of 10~ 4 M per vial) or ascorbic acid (1.7 X 10~ 4 Ai), the proportion of DMSO-water was always kept constant. TLC. The solutions were spotted on TLC plates according to the method of Stubblefield et al. (1970) and compared with known standards. Plates 20 X 20 cm were coated with 10 g of Adsorbosil (Applied Scientific Laboratories) dissolved in 11 ml of water. The plates, activated by heating for 2 hr, were spotted with 10-;ul lots of the solutions and placed in a Chromatographie chamber that contained a freshly prepared mixture of chloroform : acetone : water (88:12:1.5). The mobile phase ran through the plate in approximately 60 min, after which the plate was dried under a hood. The spots were visualized and photographs taken under ultraviolet light.
RESULTS Studies of Carcinogenesis In rats killed 5 days after the last dose of aflatoxin, histopathologic analysis, as well as enzymic determination of glucose-6-phosphatase and acid phosphatase, revealed that the livers of treated rats had undergone the characteristic early changes observed after aflatoxin exposure. There was no difference between irradiated and unirradiated animals. Enzymic assays revealed an increase of more than 100% in the activity of glucose-6-phosphatase in the aflatoxin-treated animals, despite whether they had been exposed to light. These results were consistent with the histochemical determination that indicated higher levels of glucose-6phosphatase than in controls (Table 1). There was no statistically significant difference between the concentrations of the lysosomal enzyme acid phosphatase in control and treated animals. The total enzyme activity (both free and bound) was slightly decreased in both aflatoxin groups. Although the value of bound acid
358
P. I. JOSEPH-BRAVO ET AL.
TABLE 1. Effect of Aflatoxin on Glucose-6-Phosphatase Activity in Livers of Irradiated and Unirradiated Rats
Treatment0
Glucose-6-phosphatase (nmol/mg protein)
DMSO Aflatoxin Aflatoxin + artificial sunlight
164 ± 0.039 352 ± 46.62 6 309.1 ± 6.8C
Downloaded by [New York University] at 11:40 02 June 2015
"Rats received 25 Mg of aflatoxin B, in 0.2 ml DMSO for 3 wk and were sacrificed 5 days after the last dose. The animals exposed to light were irradiated for 2 hr, 30 min after each dose. b p
Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Some interactions of light, riboflavin, and aflatoxin B1 in vivo and in vitro a
a
Patricia I. Joseph‐Bravo , Margaret Findley & Paul M. Newberne
b
a
Department of Nutrition and Food Science , Massachusetts Institute of Technology , Cambridge, Massachusetts b
Department of Nutrition and Food Science , Massachusetts Institute of Technology , Cambridge, Massachusetts, 02139 Published online: 20 Oct 2009.
To cite this article: Patricia I. Joseph‐Bravo , Margaret Findley & Paul M. Newberne (1976) Some interactions of light, riboflavin, and aflatoxin B1 in vivo and in vitro , Journal of Toxicology and Environmental Health: Current Issues, 1:3, 353-376, DOI: 10.1080/15287397609529336 To link to this article: http://dx.doi.org/10.1080/15287397609529336
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
SOME INTERACTIONS OF LIGHT, RIBOFLAVIN, AND AFLATOXIN B1 IN VIVO AND IN VITRO Patricia I. Joseph-Bravo, Margaret Findley, Paul M. Newberne
Downloaded by [New York University] at 11:40 02 June 2015
Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts In previous studies, artificial sunlight and riboflavin synerglstically increased acute aflatoxin toxIcity in rats. Three new experiments were designed to provide information on the interaction of riboflavin, aflatoxin, and light. In a study of carcInogenesis, rats received low levels of aflatoxin 5 days/wk for 3 wk; 30 min after each dosing, half of them were Irradiated for 2 hr. In some, levels of glucose-6-phosphatase and acid phosphatase were determined 5 days after completion of treatment. Remaining rats were killed at 30 or 53 wk. All underwent complete necropsies and histopathologic examination. In the second experiment, rats were dosed with riboflavin and divided into four groups: no further treatment; aflatoxin (LD50); irradiation (1-2 hr); or aflatoxin plus irradiation. Blood riboflavin levels were determined at intervals following these treatments. In the third experiment, the chemical reactions of irradiated aflatoxin and/or riboflavin were studied by uv spectroscopy and TLC. The 53-wk study showed clearly that light decreased the incidence of aflatoxin-induced cancer. The other results may provide an explanation. Aflatoxin caused blood riboflavin levels to decrease-an effect enhanced by irradiation, suggesting that photosensitized riboflavin and aflatoxin form a complex. This interpretation gains support from studies in vitro that showed that riboflavin quenched aflatoxin photodegradation, perhaps by complexing with aflatoxin. Thus, low, carcinogenic doses of aflatoxin may complex with endogenous, photosensitized riboflavin, inhibiting its degradation into carcinogenic metabolites.
INTRODUCTION In 1964, Epstein et al. described a correlation between the photodynamic and carcinogenic activities of polycyclic compounds. Aflatoxin is a photosensitive compound; light, either directly or through the agency of such light-sensitive substances as carotenes or riboflavin, is known to influence the effects of aflatoxin in vivo (Neely et al., 1970; Smith and Neely, 1972). Previous studies (Newberne et al., 1974) have shown that artificial sunlight increases the toxicity of aflatoxin Bj in rats and that light plus excess orally administered riboflavin synergistically increase toxicity. Although aflatoxin occurs in relatively high concentrations in tropical and subtropical countries, exposed populations do not all respond the same way. Other environmental factors, such as diet (e.g., dietary These studies were supported by USPHS grants ES 00616 and ES 00497 and by a grant from the Duro-Test Corporation. Requests for reprints should be sent to Paul M. Newberne, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. 353 Journal of Toxicology and Environmental Health, 1:353-376, 1976 Copyright © 1976 by Hemisphere Publishing Corporation
Downloaded by [New York University] at 11:40 02 June 2015
354
P. I. JOSEPH-BRAVO ET AL.
riboflavin levels) and exposure to sunlight, may be important in determining biologic response. We have conducted a group of experiments designed to develop new information about the interactions among aflatoxin, riboflavin, and light in the living organism. The first experiment was a study of the effects of light on aflatoxin carcinogenicity in rats. Since some liver enzymes are believed to take part in the catabolism of aflatoxin and, perhaps, the formation of carcinogenic metabolites (Allison, 1966; Danilov et al., 1970), activities of glucose-6phosphatase and acid phosphatase were examined in some rats. In the second experiment, the effects of aflatoxin and light on blood levels of riboflavin-administered rats were measured. Whether light influences the metabolism of riboflavin was questioned by Maslenikova (1962), who studied the effect of solar radiation on the levels of riboflavin in blood, organs, and urine of rats receiving 20 jug vitamin/day; urinary excretion of riboflavin was increased and the hemoglobin concentration was decreased. Ultraviolet irradiation also caused a decrease in the levels of riboflavin in the blood of animals dosed with it. Similarly, X-rays increased riboflavin excretion by rats. Correlations between levels of riboflavin and the carcinogenicity of some compounds have been reported (Rivlin, 1973). Studies have indicated that excess riboflavin protects against, and deficiency potentiates, the hepatic carcinogenicity of 4-dimethylazobenzene (DAB) (Kensler et al., 1941; Lambooy, 1970; Mulay and O'Gara, 1968). On the other hand, Rubenchik (1963) has reported that DAB intensifies the excretion of riboflavin, an effect apparently due to an increase in toxicity of nucleotide phosphatase (Yang and Sung, 1966). Riboflavin deficiency affects the microsomal oxidative and reductive pathways (Shargel and Mazel, 1973), but riboflavin treatment of well-nourished rats produced no significant alterations in microsomal enzyme activities (Gillette et al., 1972), perhaps because much of the. riboflavin injected ¡ntraperitoneally in rats is destroyed or rapidly eliminated (Bessey et al., 1958; Christensen, 1969). The formation of a complex between riboflavin and other compounds has been observed by Radda (1966). Tung and Lin (1964) have observed a quenching effect of two azo dyes, DAB and 4-aminobenzene, on riboflavin fluorescence and have proposed the formation of a complex between these drugs and riboflavin in aqueous solution. This suggested that a direct complexing may block the action of the toxin at tissue receptor sites and thus modify biologic response to the toxin. In a third experiment, the chemical interactions of aflatoxin, riboflavin, and light were studied in vitro. Having long suspected that the metabolites responsible for aflatoxin's toxicity and carcinogenicity are different, we reasoned that analysis of reaction products might indicate whether metabolites vary under different environmental conditions, such as excess riboflavin and exposure to light.
Downloaded by [New York University] at 11:40 02 June 2015
LIGHT, RIBOFLAVIN, AND AFLATOXIN B,
355
Using ultraviolet spectroscopy and thin-layer chromatography (TLC), we examined irradiated and unirradiated samples of aflatoxin, riboflavin, and aflatoxin-riboflavin mixtures. Methanol was one of the solvents used. Because we wished to produce conditions similar to those found in vivo, we also studied the reaction in the presence of water. Since aflatoxin is not sufficiently soluble in water alone, a mixture of dimethylsulfoxide (DMSO) and water was used. Song et al. (1972) have reported that potassium iodide at concentrations of 10~ 4 M quenches the photoreduction of riboflavin when riboflavin is in the excited triplet state. This results from the formation of a strong complex between the alloxazine moiety of riboflavin and iodide in a proportion of 1:1. To determine whether this was the case in our system, we evaluated the influence of potassium iodide on the photoreduction of riboflavin in the riboflavin-aflatoxin mixture. The influence of a known inhibitor of riboflavin photoreduction, ascorbic acid (Reusser, 1970), was also examined. As a final check on the importance of the solvent used, aflatoxin and the aflatoxin-riboflavin mixture were dissolved in chloroform, irradiated, and subjected to TLC. METHODS Studies of Carcinogenesis Sixty male weanling rats of the Charles River CD strain (SpragueDawley, Charles River Breeding Laboratories) were housed in stainless steel cages and supplied with tap water ad libitum. Their weights were recorded periodically. All animal rooms were kept at 72°F and under controlled lighting (12-hr cycle). The complete, semisynthetic diet used throughout the experiment (Rogers and Newberne, 1971) was fed for 1 wk prior to initiation of aflatoxin treatment. Each rat was dosed intragastrically with 25 /ig of aflatoxin B r (Makor Chemicals, Jerusalem, Israel) in 0.2 ml DMSO (Fischer Scientific), 5 days/wk for 3 wk, to receive a total carcinogenic dose of 375 jug. Control animals received 0.2 ml DMSO. Thirty minutes after each aflatoxin administration, half of the rats from each group were irradiated for 2 hr with a long-arc xenon lamp (Vita-Lite, Duro-Test Corp.) with a filter combination giving a spectral distribution in the ultraviolet and visible ranges closely approximating natural light (Newberne et al., 1974). To attenuate the strong infrared resonance line of xenon, a water cell of plastic material (approximately 5 cm) was placed 50 cm below the lamp. Five days after the last aflatoxin dose was given, five rats from each group were decapitated, complete necropsies performed, and organs (liver, kidney, spleen, stomach, testes, heart, lung, and thymus) removed for histopathologic evaluation. Samples of liver were prepared in one of three
Downloaded by [New York University] at 11:40 02 June 2015
356
P. I. JOSEPH-BRAVO ET AL.
ways: fixed in formalin; frozen in liquid nitrogen for biochemical analysis of glucose-6-phosphatase activity and for histochemical determination of glucose-6-phosphatase and acid phosphatase; or used for immediate determination of acid phosphatase activity. Tissue concentration of acid phosphatase was determined by the method described in Sigma Technical Bulletin No. 104 (1968). For histochemical assay, the Gomori method was used (Barka and Anderson, 1965). The activity of glucose-6-phosphatase was determined according to the method of Harper (1965). For histochemical assay, the method of Wachstein and Merzel (1956) was employed. All reagents were purchased from the Sigma Chemical Company. Thirty weeks after the last aflatoxin dose, 10 rats from each group were decapitated, necropsies were performed, and organs and tissues removed for histopathologic examination. The remaining rats were sacrificed and studied 53 wk after aflatoxin treatment. Any rat that died during the 53-wk period was autopsied and its tissues examined histologically. Effect of Light and Aflatoxin on Riboflavin Levels The housing conditions, diet, and strain of rat used were the same as described above. On the day of the experiment, riboflavin-water solutions were prepared under dim light; the vitamin was dissolved in tubes covered with aluminum foil. The rats were dosed intragastrically with 1 or 10 mg riboflavin (General Biochemicals) per kilogram body weight, and riboflavin levels were measured in blood at appropriate intervals. The effect of aflatoxin on riboflavin levels was studied in 10 rats that received an L D 5 0 of aflatoxin (5 mg/kg body weight; Makor Chemicals, Jerusalem, Israel) in 0.2 ml DMSO, 45 min after they had received the riboflavin. Half the rats were irradiated for 1-2 hr. Rats were anesthetized with ether and bled by heart puncture. Riboflavin levels were determined in whole blood with an Aminco fluoromicrophotometer; the method of Clarke (1969), modified to use 6 ml of blood instead of 12 ml, was employed. Chemical Reactions in Vitro Methanol as solvent. Aflatoxin B! (Makor Chemicals) was dissolved in methanol (Allied Chemical Corp.), to a concentration of 1.78 X 10~ 4 M, in scintillation vials covered with aluminum foil. This concentration was chosen according to the extinction coefficient (approximately 2,000) to yield an absorbance value greater than 1. Because we wished to observe a 1:1 interaction, riboflavin (General Biochemicals) was dissolved under the same conditions to give a final concentration of 1.78 X 10~ 4 M. The solutions were placed under the irradiation source (as described above) in
Downloaded by [New York University] at 11:40 02 June 2015
LIGHT, RIBOFLAVIN, AND AFLATOXIN B,
357
uncovered vials for periods ranging from 15 min to 5 hr. When aflatoxin was irradiated in the presence of riboflavin, care was taken to have the same final concentration of 1.78 X 10~ 4 M. At the end of the irradiation, the vials were covered with aluminum foil and caps, and the spectrum was recorded immediately in a Gilford spectrophotometer 240, or spotted on TLC plates. DMSO-water as so/vent. Because DMSO is present in the blood of rats 2 hr after its administration as a solvent for aflatoxin (Newberne et al., 1974), the lowest proportion of DMSO to water possible without precipitation was used. Aflatoxin was dissolved first in the DMSO (Fischer Scientific), to which water was gradually added to give a final concentration of 10% (v/v). In the riboflavin solutions, DMSO was added in the same proportions. When the mixture was irradiated in the presence of potassium iodide (at a concentration of 10~ 4 M per vial) or ascorbic acid (1.7 X 10~ 4 Ai), the proportion of DMSO-water was always kept constant. TLC. The solutions were spotted on TLC plates according to the method of Stubblefield et al. (1970) and compared with known standards. Plates 20 X 20 cm were coated with 10 g of Adsorbosil (Applied Scientific Laboratories) dissolved in 11 ml of water. The plates, activated by heating for 2 hr, were spotted with 10-;ul lots of the solutions and placed in a Chromatographie chamber that contained a freshly prepared mixture of chloroform : acetone : water (88:12:1.5). The mobile phase ran through the plate in approximately 60 min, after which the plate was dried under a hood. The spots were visualized and photographs taken under ultraviolet light.
RESULTS Studies of Carcinogenesis In rats killed 5 days after the last dose of aflatoxin, histopathologic analysis, as well as enzymic determination of glucose-6-phosphatase and acid phosphatase, revealed that the livers of treated rats had undergone the characteristic early changes observed after aflatoxin exposure. There was no difference between irradiated and unirradiated animals. Enzymic assays revealed an increase of more than 100% in the activity of glucose-6-phosphatase in the aflatoxin-treated animals, despite whether they had been exposed to light. These results were consistent with the histochemical determination that indicated higher levels of glucose-6phosphatase than in controls (Table 1). There was no statistically significant difference between the concentrations of the lysosomal enzyme acid phosphatase in control and treated animals. The total enzyme activity (both free and bound) was slightly decreased in both aflatoxin groups. Although the value of bound acid
358
P. I. JOSEPH-BRAVO ET AL.
TABLE 1. Effect of Aflatoxin on Glucose-6-Phosphatase Activity in Livers of Irradiated and Unirradiated Rats
Treatment0
Glucose-6-phosphatase (nmol/mg protein)
DMSO Aflatoxin Aflatoxin + artificial sunlight
164 ± 0.039 352 ± 46.62 6 309.1 ± 6.8C
Downloaded by [New York University] at 11:40 02 June 2015
"Rats received 25 Mg of aflatoxin B, in 0.2 ml DMSO for 3 wk and were sacrificed 5 days after the last dose. The animals exposed to light were irradiated for 2 hr, 30 min after each dose. b p
