Effects of riboflavin deficiency and riboflavin administration on carcinogen-DNA binding

Fd Chem. Toxic. Vol. 31, No. 10, pp. 745-750, 1993 Printed in Great Britain. All rights reserved

0278-6915/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd

EFFECTS OF RIBOFLAVIN DEFICIENCY A N D RIBOFLAVIN ADMINISTRATION ON C A R C I N O G E N - D N A BINDING J. PANGREKAR, K. KRISHNASWAMY and V. JAGADEESAN* Food and Drug Toxicology Research Centre, National Institute of Nutrition, Jamai-Osmania, Hyderabad 500 007, India

(Accepted 20 April 1993) Abstract--A study was conducted to assess the effects of ribofiavin deficiency and riboflavin supplementation on carcinogen-DNA binding. After 12 wk on a riboflavin-sufficient or a riboflavin-deficient diet male Wistar rats were administered 3H-labelled benzo[a]pyrene (BP) ip. [3H]BP was given either at a uniform dose of 450 #Ci/rat irrespective of body weight or at a dose adjusted to body weight. After 17 hr the animals were killed, various organs were dissected and the level of [3H]BP bound to DNA was quantified in organs that are known to be the seats of drug metabolism (i.e. the liver, lungs and intestinal mucosa). In a separate experiment, the effect of riboflavin supplementation on BP-DNA binding was also investigated. When [3H]BP was administered at 450 #Ci/rat, BP-DNA binding was markedly increased in the livers and intestinal mucosae of the pair-fed and deficient groups compared with controls. With the administration of [3H]BP adjusted to body weight, no differences in BP-DNA binding between groups were observed in any tissue. However, on administration of riboflavin there was a decrease in the level of [3H]BP bound to DNA in almost all tissues, especially in the lungs, where the reduction was significant. The results suggest that undernutrition/riboflavin deficiency may increase the risk of carcinogenesis by way of an increase in carcinogen binding, which however can be reversed by riboflavin supplementation.

INTRODUCTION

MATERIALS AND METHODS

Riboflavin deficiency is c o m m o n in m a n y parts o f the developing world (Bamji, 1988; Bates, 1987). A p a r t from various clinical manifestations, such as angular stomatitis and cheilosis, riboflavin deficiency is implicated in physiological functions like lipid peroxidation, energy processes, etc. Recently, riboflavin deficiency has also been reported to be a risk factor in oesophageal cancer ( T h u r n h a m et al., 1985; Van Rensburg et al., 1983). In view o f the fact that more than 70-80% o f h u m a n cancers are now ascribed to environmental factors, and 30% o f these cancers are o f dietary origin (Doll and Peto, 1981), the present study was carried out to investigate the mechanisms of chemical carcinogenesis in riboflavin deficiency. Carcinogen D N A binding, which has been suggested as a necessary event in chemical induction o f tumours, was investigated in the normal (control) and riboflavin-deficient states. In addition, the beneficial effect o f riboflavin supplementation, if any, was also assessed.

*To whom correspondence should be addressed. Abbreviations: BP = benzo[a]pyrene; EGRAC = erythrocyte glutathione-reductase activity coemcient; FAD = flavin adenine dinucleotide; FMN = flavin mononucleotide; FR = free riboflavin; GSSG = glutathione oxidized form; RNase = ribonuclease; SSC buffer = 0.15 M-sodium citrate-sodium chloride buffer. 745

Chemicals Tritium-labelled benzo[a]pyrene (BP; sp. act. 3 0 C i / m m o l ) was obtained from Radiochemical Centre (Amersham, Bucks., UK). Triton X-100, ribonuclease (RNase), protease K, calf thymus D N A , N A D P H , flavin adenin dinucleotide ( F A D ) and oxidized glutathione (GSSG) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Soluene-100 was obtained from Packard Instrument Co., Inc. (Downers Grove, IL, USA). All other chemicals were o f analytical grade. Table 1. Composition of the diets given to male Wistar rats in the control, riboflavin-deficientand pair-fed groups* Content Ingredient (g/100 g diet) Sucrose 35.0 Starch 35.0 Vitamin-free casein 20.0 Groundnut oil 5.0 Salt mixture 4.0 Vitamin mixture+ 0.6 Choline chloride mixture';" 0.4 *Riboflavin was added to the control diet only to provide a level of 9.5 mg/kg diet. "l'ln addition to water-soluble vitamins, vitamin A, vitamin D and vitamin E dissolved in groundnut oil were also added to the diet at the recommended levels. d:~Choline chloride mixture= 50g choline chloride + 150 g starch.

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J. PANGREKARet al.

Control (28)

Pairfed (28)

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Deficient (28)

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Riboflavin injected (CR)

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(14)

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Fig. 1. Diagrammatic representation of the distribution of rats in different groups in experiment 2. The numbers in parentheses indicate the numbers of rats used. Experimental protocol Experiment 1. The first experiment consisted of administering to rats radiolabelled BP at a uniform dose of 450 pCi/animal irrespective of body weight. For this purpose, 32 weanling male Wistar rats (Wistar/NIN strain, 30-50g body weight) were divided randomly into three groups: control (10), riboflavin deficient (12) and pair-fed (10). The animals were maintained under the following conditions: temperature, 2 2 + I°C; humidity, 55%; and 12-hr light/dark cycles. They were housed individually and had free access to water. They were fed on their respective diets as detailed in Table 1. Riboflavin was omitted from the vitamin mixture of the deficient diet. The riboflavin content of the control diet was 9.5 mg/kg diet and that of the deficient diet was below 0.035mg/kg diet. Control and riboflavin-deficient rats received their respective diets ad lib. Since it is

known that food consumption is reduced in deficient animals, the third group consisting of pair-fed rats, was given the control diet at an amount equivalent to that consumed by the riboflavin-deficient group. F o o d consumption was recorded daily, and body weights were measured weekly. Erythrocyte glutathione-reductase activity coefficient ( E G R A C ) (Tillotson, 1971) and liver riboflavin levels (Bessey et al., 1949) were estimated to monitor riboflavin deficiency. After 12 wk the rats were administered ip 450/~Ci [3H]BP (equivalent to 4/~gBP) in corn oil. The injection was always given around 5 pm, and the animals were killed on the next day at 10am (i.e. 17 hr after BP administration). The liver, lungs and small intestine (25 cm of the proximal portion), which are known to be the sites of xenobiotic metabolism, were quickly removed and processed for the isolation of radioactivity bound to D N A (Essigmann et al., 1977). Briefly, the procedure consisted of: isolation of

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Week Fig. 2. Growth curves and mean body weight gains of control, pair-fed and riboflavin-deficient rats on their respective diets for a period of 12 wk.

Riboflavin deficiency and carcinogen-DNA binding

747

Table 2. Nutritional status of riboflavin of rats in the control and experimental groups Group

Control

Riboflavindeficient

Pair-fed

1.2 +_ 0.11 a (5) 22.5 ± 2.72a (5)

1.9 ± 0.16 ab (6) 12.5 ± 2.71 ab (6)

1.3 ± 0.17 b (5) 24.5 _+ 1.67 b (5)

Parameter Erythrocyte glutathione-reductase activity coefficient Liver riboflavin content (/~g/g liver)

Values are means + SEM of the numbers of rats indicated in parentheses. Values bearing the same superscript are significantly different (P <0.001: horizontal comparisons).

nuclei, proteinase K and RNase treatments, repeated extractions with chloroform-isoamyl alcohol (24:1, v/v) and precipitation with ice-cold alcohol. The DNA precipitate was dried, dissolved in 0.15 Msodium citrate-sodium chloride (SSC) buffer and subjected to RNase and protease K treatments. Extraction and precipitation were then repeated, and the DNA was washed and dissolved in SSC buffer. Part of this solution was used for liquid scintillation counting, while DNA content was estimated from the remainder by diphenylamine reaction. The results were expressed as fmol BP bound/mg DNA. Statistical analysis was done by one-way analysis of variance. Means were tested by least-significant differences. A statistical difference at P < 0.05 was considered to be significant. Experiment 2. In the second study, radiolabelled BP was administered to rats at a dose adjusted to body weight; any possible beneficial effect of riboflavin administration prior to BP treatment was also investigated. 84 male weanling Wistar rats (NIN/Wistar strain, 4 5 ~ 0 g body weight) were divided randomly into three groups of 28 animals each: control, riboflavin deficient and pair-fed. The rats were maintained under the same conditions as for experiment 1. All animals were fed on their respective diets for a period of 12wk. After the end of the experimental period, each of the three groups was divided equally into two sub-groups: rats in the first sub-group received ip 5mg riboflavin/100g body

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weight in saline, and those in the second sub-group were treated with saline only (Fig. 1). 24hr after administration of riboflavin/saline, all animals were injected ip 113/~ci BP/100 g body weight in corn oil. The rats were killed 17 hr after BP injection, and tissues were quickly removed and processed for the radioactivity bound to DNA (expressed as fmol BP bound/mg DNA) using the procedure described above. RESULTS

Body weight gain and food consumption Body weight gains in the control, pair-fed and riboflavin-deficient groups during the experimental period are shown in Fig. 2. There was a marked reduction in body weight gain in the riboflavindeficient group compared with controls. This decrease was due to large differences in food intake between the two groups; rats in the control group consumed on average 15 g food per day, whereas food intake was only about 6g per day in the riboflavin-deficient group. A decrease in body weight gain was also observed in the pair-fed group since the daily amount of food given to rats in this group was restricted to match daily food consumption in the riboflavin-deficient group.

Markers of riboflavin deficienO, Table 2 shows the data on EGRAC and liver riboflavin levels; these parameters are generally adopted as markers of riboflavin deficiency. The occurrence of riboflavin deficiency was confirmed by an increase in EGRAC and a decrease in liver riboflavin levels. These parameters were normal in the pair-fed group. Clinical signs of riboflavin deficiency, such as scaling of hair and areas of alopecia, were observed in the riboflavin-deficient group.

BP-DNA binding When [3H]BP was administered at a uniform level

iiiiiii!iliiiiiiii of 450pCi/rat, binding to DNA was found to be !i!iii~i!i!~!~!i Liver

Lung

IM

Fig. 3. Binding of [3H]benzo[a]pyrene(450 pCi/rat)to DNA in the livers, lungs and intestinal mucosa (IM) of rats in the control, riboflavin-deficientand pair-fed groups in experiment 1. Values are means+ SEM of 10 (control), 12 (riboflavin-deficient)and 10 (pair-fed) rats. Values bearing the same superscript are significantly different (P < 0.05).

significantly higher in the liver, both in riboflavindeficient and pair-fed animals, as compared with controls (Fig. 3). The bound radioactivity in the liver was 0.62, 2.36 and 1.79 fmol/mg DNA in the control, riboflavin-deficient and pair-fed groups, respectively. However, between the pair-fed and riboflavindeficient groups no significant differences were noted

J. PANGREKAR el

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al.

Table 3. Effect of riboflavin injection on benzo[a]pyrene (BP)-DNA binding (expressed as fmol BP/mg DNA) in various tissues of rats in the control, pair-fed and riboflavin-deficient groups Tissue Liver Lungs Intestinal mucosa

Control + saline

Control + riboflavin

0.37 ± 0.109 (7) 0.40 _+0.045 (7) 0.47+0.117 (7)

0.52± 0.042 (7) 0.40 ± 0.048 (7) 0.52 ± 0.120 (7)

Pair-fed+ saline

Pair-fed+ riboflavin

Deficient+ saline

0.30± 0.054 0.29± 0.069 0.29 ± 0.121 (6) (6) (5) 0.27 ± 0.063 0.19 ± 0.029 0.45 ± 0.082b (7) (7) (6) 0.87_+0.366 0.35_+0.058 0.24_+0.035 (7) (7) (6)

Deficient+ riboflavin 0.26 ± 0.044 (6) 0.23 ± 0.058b (6) 0.27+0.078 (6)

The BP dose used was: 113 #Ci/100 g body weight. Values are means ± SEM of the numbers of rats indicated in parentheses. Values with the same superscripts are significantly different (P < 0.05; horizontal comparisons).

a l t h o u g h there w a s an increasing t r e n d in the latter g r o u p . T h e b i n d i n g o f BP to D N A in the intestinal m u c o s a t I M ) also s h o w e d a similar p a t t e r n , w i t h significantly h i g h e r B P - D N A b i n d i n g in b o t h the riboflavin-deficient a n d pair-fed g r o u p s . N o signific a n t c h a n g e s were o b s e r v e d in the l u n g s b e t w e e n g r o u p s . B P - D N A b i n d i n g in the tissues followed a similar p a t t e r n in all the three g r o u p s a n d w a s in the order: I M > liver > lungs. In the s e c o n d study, BP w a s a d m i n i s t e r e d at a d o s e a d j u s t e d to b o d y weight. T h e results o f this s t u d y s h o w e d t h a t there w a s n o significant difference in B P - D N A b i n d i n g in tissues b e t w e e n g r o u p s ( T a b l e 3). T h i s is in c o n t r a s t to the results o f experi m e n t 1, w h e r e a n increased B P - D N A b i n d i n g w a s n o t e d in riboflavin-deficient rats.

Effect of riboflav& admin&tration R i b o f l a v i n a d m i n i s t r a t i o n did n o t have a n y signific a n t effect o n b o d y w e i g h t s a n d o r g a n w e i g h t s o f a n i m a l s in all the three g r o u p s . E G R A C , w h i c h w a s increased significantly in riboflavin-deficient rats, w a s n o r m a l i z e d by riboflavin a d m i n i s t r a t i o n w i t h i n 41 hr. R i b o f l a v i n a d m i n i s t r a t i o n increased the levels o f F A D , F M N a n d free riboflavin ( F R ) in all the tissues studied in the c o n t r o l , pair-fed a n d riboflavindeficient g r o u p s ( T a b l e 4).

T h e effect o f riboflavin a d m i n i s t r a t i o n on the perc e n t a g e o f total radioactivity p r e s e n t in several tissues w a s initially assessed. A r e d u c t i o n in radioactivity, s o m e t i m e s significant, w a s o b s e r v e d in a l m o s t all the o r g a n s studied (Fig. 4A,B); this was n o t e d in b o t h the pair-fed c o n t r o l a n d riboflavin-deficient g r o u p s . Subsequently, B P - D N A b i n d i n g w a s quantified only in o r g a n s t h a t are i m p o r t a n t f r o m the p o i n t o f view o f d r u g m e t a b o l i s m (i.e. the liver, lungs a n d intestinal mucosa). T h e a d m i n i s t r a t i o n o f riboflavin caused m a r k e d c h a n g e s in B P - D N A b i n d i n g (Table 3; Fig. 5). A f t c r riboflavin a d m i n i s t r a t i o n , there w a s a significant red u c t i o n in B P - D N A b i n d i n g in the lungs, a target o r g a n for BP, in riboflavin-deficient rats (0.45 t:. 0.23 fmol). Similarly, a 30% r e d u c t i o n in b i n d i n g in the l u n g tissues a n d a m a r k e d 6 0 % r e d u c t i o n in the I M were o b s e r v e d in the pair-fed g r o u p . S o m e o f thc o t h e r c h a n g e s were m a r g i n a l ; h o w e v e r , they indicated a decreasing t r e n d in B P - D N A b i n d i n g on riboflavin a d m i n i s t r a t i o n . A n o n - s i g n i f i c a n t small increase in D N A b i n d i n g in the liver w a s o b s e r v e d in the c o n t r o l g r o u p on riboflavin s u p p l e m e n t a t i o n . DISCUSSION Deficiencies in m i c r o n u t r i e n t s such as selenium. zinc a n d riboflavin are implicated in the c a u s a t i o n o f

Table 4. Effects of riboflavin deficiency and riboflavin supplementation on body weights, hepatic erythrocyte glutathione-reductase activity coefficients (EGRAC) and total flavin levels of rats

Body weight (g) Before saline or riboflavin injection After saline or riboflavin injection EGRAC Before saline or riboflavin injection After saline or riboflavin injection Total flavinst (t*g/g tissue) Liver Lungs Intestinal mucosa

Control + saline

Control + riboflavin

Pair-fed+ saline

Pair-fed+ riboflavin

Deficient + saline

Deficient riboflavin

355 ± 6.9~b (7) 347 ± 7.8ab (7)

358 + 5.9 (7) 345 ± 5.1 (7)

124 ± 1.7~ (7) 122 + 2.0ac (7)

122 ± 2.4 (7) 121 ± 2.2 (7)

66 + 2.7~ (6) 64 + 3.5~ (6)

79 ~ 12 I (6) 74 + 12. [ !61

1.2 ± 0.04~ (7) 1.1 _+0.04~ (7)

1.2 _+0.03 (7) hi ±0.03 (7)

1.1 + 0.04b (7) 1.2 ±0.02 b (7)

1.2 +_0.05 (6) I.I +0.04 (7)

1.95 + 0.12"' (6) 1.8 _+0.16~l' (6)

I 75 + 007* (6) I I ~ 0.04" (6)

28.4 + 0.86~d (6) 3.1 _+0.19" (6) 11.2_+0.63" (6)

41.8± 2.05~ (6) 9.1 ± 0.58" (6) 68.7± 1.7~ (6)

26.9 ± 1.27~ (6) 3.1 _+0.208 (6) 12.3_+0.36~¢ (6)

44.1 ± 2.25b (6) 16.2 ± 1 . 3 2 (6) 42.9± 1.75b (6)

13.3 ± 0.94~d~ (5) 2.9+0.11" (5) 8.2_+0.25~a~ (5)

27.6 + 0.45 15) 6.1 + 0.35' (5) 24.1 + 1.16~ (5/

Values are means + SEM of the numbers of rats indicated in parentheses. Values with the same superscripts are significantly different (P < 0.01; horizontal comparisons). *P < 0.001 ; vertical comparisons. "tTotal flavins = FAD + FMN + FR.

Riboflavin deficiency and carcinogen-DNA binding 18

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Fig. 4. Proportion of the dose of [3H]benzo[a]pyrene administered (113/~Ci/100 g body weight) m various tissues of rats in experiment 2: CS = control + saline; CR = control + riboflavin; PS = pair-fed + saline; PR = pair-fed + riboflavin; DS = riboflavin-deficient + saline; DR = riboflavin-deficient + riboflavin. m

160 ~ Liver

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140 IM

120 100

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80 60 40 Fig. 5. Effect of riboflavin administration on the binding of [3H]benzo[a ]pyrene (l 13 # Ci/100 g body weigh t) to DNA in the livers, lungs and intestinal mucosae (IM) of rats in experiment 2: CR = control + riboflavin; PR = pairfed + riboflavin; DR = riboflavin-deficient + riboflavin. The values obtained from rats untreated with riboflavin were taken as 100%.

h u m a n cancers; h o w e v e r , their exact m e c h a n i s m s are not completely understood. There is clear evidence showing that chemicals and environmental pollutants are involved in the aetio]ogy o f h u m a n cancers. The damage to D N A caused by the binding o f reactive metabolites, which are generated by biotransformation o f carcinogenic c o m p o u n d s , is one o f the i m p o r t a n t mechanisms suggested. Our earlier i n v e s t i g a t i o n s have shown that under nutrient deficiencies the activities o f enzymes involved in carcinogen metabolism are altered (Jagadeesan, 1989; Ramesh and K r i s b n a s w a m y 1985). We have also demonstrated increased c a r c i n o g e n - D N A binding under food restriction (Jagadeesan and Krishnaswamy, 1989) in laboratory animals. All these studies indicate that under nutrient deficiency enzymatic metabolic activation and D N A binding o f carcinogens may be the critical steps in carcinogenesis; hence the latter mechanism was chosen as an endpoint to investigate riboflavin deficiency in detail.

750

J. PANGREKAR et al.

In o u r first experiment, a uniform dose of 450/~Ci BP per rat was used since h u m a n exposure to e n v i r o n m e n t a l a n d other pollutants occurs independently o f body mass, size or weight. The results unequivocally suggested higher binding in riboflavindeficient animals, which is in line with our earlier observations (Jagadeesan and Krishnaswamy, 1985), Since pharmacological assessments are made on a dose-to-body weight basis, the second phase of the study involved a d m i n i s t r a t i o n of the carcinogen adjusted to the body weights of animals. The effect of riboflavin was studied only in the second experiment, where the administered levels of the carcinogen a n d the vitamin were related to body weight, since o u r aim was to establish a relationship between carcinogen and riboflavin doses. The results of these studies indicated that a l t h o u g h riboflavin-deficient a n d foodrestricted rats had similar levels of binding, riboflavin a d m i n i s t r a t i o n could reduce carcinogen D N A interaction. Earlier studies in vitro by B h a t t a c h a r y a et al. (1987) a n d W o o d et al. (1982) have attributed the beneficial effect of riboflavin to F M N , F A D and FR. In the present study, riboflavin a d m i n i s t r a t i o n increased the levels of F M N , F A D a n d FR, which were generated m vivo. These results indicate that total flavins (either in the form of F R or F M N / F A D ) p r o b a b l y c a p t u r e d the reactive metabolites o f BP by forming complexes with them ( W o o d et al., 1982). The above observations clearly d e m o n s t r a t e a beneficial effect of riboflavin. A d m i n i s t r a t i o n of the vitamin seems to favour a faster removal of radioactivity in the tissues and therefore decreases carcinogen binding to cellular macromolecules like D N A , as seen in the lungs a n d IM. However, we did not a t t e m p t to identify the B P - D N A adducts in the present study, since BP metabolism a n d a d d u c t i o n to cellular macromolecules are well k n o w n and have been extensively studied (Conney, 1982). Intake of riboflavin at higher than r e c o m m e n d e d doses may thus help in reducing the risk of cancer in p o p u l a t i o n s with deficiency in this vitamin. In the present study, rats were given 50 m g riboflavin/kg body weight since this dose has been shown to elevate the levels of pyridine nucleotides within 48 hr. Experiments with lower doses of riboflavin are under way in order to establish the m i n i m u m dose required that produces the effects observed in the present study.

REFERENCES

Bamji M. S. (1988) Biochemical detection, aetiology and functional consequences of riboflavin deficiency. Proceedings of the Indian National Science Academy 1, 1~6. Bates C. J. (1987) Human riboflavin requirements and metabolic consequences of deficiency in man and animals. World Review of Nutrition Dietetics 50, 215 265. Bessey O. H., Lowry O. H. and Love R. H. (1949) The fluorometric measurement of the nucleotides of riboflavin and their concentration in tissues. Journal o/" Biological Chemistry 180, 755 769. Bhattacharya R. K., Francis A. R. and Shetty T. K. (1987) Modifying role of dietary factors on the mutagenicity of aflatoxin B~. In vitro effect of vitamins. Mutation Research 188, 121-128. Conney A. H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic hydrocarbons. Cancer Research 42, 4875 J,917. Doll R. and Peto R. (1981) The causes of cancer. Quantitative estimates of avoidable risk of cancer in the United States today. Journal q[" the National Cancer Institute 66, 1191 1308. Essigmann J. M., Croy R. G., Nadzan A. M., Busby W F., Jr, Reinhold V. N., Buchi G. and Wogan G. N. (1977) Structural identification of major DNA adduct formed by AFB I in vitro. Proceedings of the National Academy o f Sciences of the U.S.A. 74, 1870 1874. Jagadeesan V. (1989) Study of activating and conjugating enzymes of drug metabolism in zinc deficiency. Indian Journal o f Experimental Biology 27, 799 801. Jagadeesan V. and Krishnaswamy K. (1989) Effect of lood restriction of benzo(a)pyrene binding to DNA in Wistar rats. Toxicology 56, 223 226. Ramesh R. and Krishnaswamy K. (1985) Effect of chronic undernutrition on glucuronide and glutathione conjugation in rat liver. Drug Nutrient blteractions 3, 121 128. Thurnham D. I., Zheng S. F., Munoz N., Crespi M., Grassi A., Hambridge M. and Chai T. F. (i985) Comparison of riboflavin, vitamin A and zinc status of Chinese populations at high and low risk for ocsophageal cancer. Nutrition and Cancer 7, 13I 143. Tillotson J. A. and Sauberlich H. E. {1971) Effect of riboflavin depletion and repletion on the erythrocyle glutathione reductase in the rat. Journal of Nutrition 101, 1459 1466. Van Rensburg S. J., Benade A. S., Du Plcssis J. P. and Du Rose E. F. (1983) Nutritional status of African populations predisposed to oesophageal cancer. Nutrition attd Cancer 4, 206- 216. Wood A. W., Sayer J. M., Newmark H. k., Yagi H., Michaud D. P., Jerina D. M. and Conney A. H (1982) Mechanism of the inhibition of mutagenicity of benzo(a)pyrene, 7,8-diol, 9,10-epoxide by riboflavin 5-phosphate. Proceedings o f the National Academy ol Science.~ ~I the U.S.A. 79, 5122 5126.