Betel nut (Areca catechu) consumption and the induction of glucose intolerance in adult CD1 mice and in their F1 and F2 offspring

Diabetologia, Jan 1994

Summary Many mutagenic nitroso compounds are also diabetogenic. Betel-nut (Areca catechu) chewing populations have an increased incidence of foregut cancers related to betel-nut nitrosamines which suggests that betel consumption could be diabetogenic. Young adult CD1 mice with a low spontaneous incidence of diabetes were fed betel nut in standard feed for 2–6 days. Single point (90 min) intra-peritoneal glucose tolerance tests were used to follow glucose tolerance up to 6 months of age. Glucose intolerance was defined as over 3 SD above mean control values. Glucose intolerance was found in 3 of 51 male and 4 of 33 female adult mice which were fed the betel diet (p<0.01). Studies on the progeny of these mice are presented separately for animals studied in Aberdeen (Group 1) and London (Group 2). In matings of Group 1 betel-fed parents glucose intolerance was found in 4 of 25 male and 1 of 22 female F1 offspring, with significant hyperglycaemia in F1 males born to hyperglycaemic but not to normoglycaemic mothers (p<0.01). In the F2 generation 4 of 23 males and 1 of 16 females and in the F3 generation 1 of 16 males and 0 of 20 females were glucose intolerant. In the Group 2 studies where betel-fed parents were mated to normal controls glucose intolerance was found in 10 of 35 male and 10 of 33 female Fl progeny (p<0.005), and mean islet areas were increased in offspring of betel-fed parents (p<0.001). The total incidence of glucose intolerance in Fl progeny from studies in Groups 1 and 2 was 14 of 60 males and 11 of 55 females (p<0.005). Insulin dependence did not develop in the glucose-intolerant betel-fed animals or their descendants; affected animals appearing well built and active. The development of glucose intolerance in F1 offspring was not dependent on maternal glucose intolerance or on maternal betel-feeding, and 90-min glucose levels of F1 offspring were directly related to paternal but not to maternal glycaemia (p<0.01). Our findings suggest that betelnut (Areca) consumption may be diabetogenic and induce an inheritable abnormality. The hypothesis is of interest in view of the widespread habit of betel consumption and of the strategies known to inhibit the induction of experimental diabetes by diabetogenic nitroso compounds.

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Betel nut (Areca catechu) consumption and the induction of glucose intolerance in adult CD1 mice and in their F1 and F2 offspring

Diabetologia Betel nut (Preca catechu) consumption and the induction of glucose intolerance in adult CD1 mice and in their F1 and F2 offspring B. J. B o u c h e r 0 1 S. W. B. E w e n 0 1 J. M. Stowers 0 1 0 Correspondingauthor:Dr. B. J. Boucher, The Medical Unit, The Royal London Hospital , Whitechapel Road, London E11BB , UK 1 1Cellular Mechanisms Research Group, London Hospital Medical College , London , UK 2Department of Pathology,Aberdeen Royal Infirmary,Aberdeen University , Aberdeen , UK Summary Many mutagenic nitroso compounds are also diabetogenic. Betel-nut (Areca catechu) chewing populations have an increased incidence of foregut cancers related to betel-nut nitrosamines which suggests that betel consumption could be diabetogenic. Young adult CD1 mice with a low spontaneous incidence of diabetes were fed betel nut in standard feed for 2~5 days. Single point (90 min) intra-peritoneal glucose tolerance tests were used to follow glucose tolerance up to 6 months of age. Glucose intolerance was defined as over 3 SD above m e a n control values. Glucose intolerance was found in 3 of 51 male and 4 of 33 female adult mice which were fed the betel diet (p < 0.01). Studies on the progeny of these mice are presented separately for animals studied in A b e r d e e n (Group 1) and London (Group2). In matings of Group 1 betel-fed parents glucose intolerance was found in 4 of 25 male and 1 of 22 female F1 offspring, with significant hyperglycaemia in F1 males born to hyperglycaemic but not to normoglycaemic mothers (p < 0.01). In the F2 generation 4 of 23 males and 1 of 16 females and in the F3 generation 1 of 16 males and 0 of 20 females were glucose intolerant. In the Group 2 studies where betel-fed parents were mated to normal controls glucose intolerance was found in 10 of 35 male and 10 of 33 female F1 progeny (p < 0.005), and mean islet areas were increased in offspring of betel-fed parents (p < 0.001). The total incidence of glucose intolerance in F1 progeny from studies in Groups 1 and 2 was 14 of 60 males and 11 of 55 females (p < 0.005). Insulin dependence did not develop in the glucose-intolerant betel-fed animals or their descendants; affected animals appearing well built and active. The development of glucose intolerance in F1 offspring was not dependent on maternal glucose intolerance or on maternal betel-feeding, and 90-min glucose levels of F1 offspring were directly related to paternal but not to maternal glycaemia (p < 0.01). Our findings suggest that betelnut (Areca) consumption may be diabetogenic and induce an inheritable abnormality. The hypothesis is of interest in view of the widespread habit of betel consumption and of the strategies known to inhibit the induction of experimental diabetes by diabetogenic nitroso compounds. [Diabetologia (1994) 37: 49-55] Diabetes mellitus; nitrosamines; nitrosocompounds; Areca catechu; Betel nut; inheritance - 9 Springer-Verlag1994 The prevalence of Type 2 (non-insulin-dependent) diabetes mellitus in Asians who have migrated from Bangladesh to the United Kingdom over the last 30 years is high, as it is in other immigrant Asian groups [ 1, 2 ]. This is not due simply to an increase in Western life styles since an equally high prevalence has been found in some groups of Asians in Fiji and in India, both in towns and in certain rural areas [ 3, 4 ]. It has also been noted that Asians with Type 2 diabetes living in the United Kingdom require insulin at a much younger age than Type 2 diabetic Caucasians [ 5 ]. The prevalence of Type 2 diabetes in the Asian immigrant population has not begun to decline towards that of the indigenous population of the United Kingdom. In addition to possible genetic factors, this community may ap < 0.01; b p < 0.001;p = NS for all other comparisons Betel +, Betel-fed (2-8 days); IGT +, impaired glucose tolerance defined as 90-min plasma glucose > 3SD above mean control levels t h e r e f o r e h a v e b r o u g h t active aetiological agents for d i a b e t e s w i t h t h e m . T h e i n c r e a s e d m o r t a l i t y f r o m o r a l a n d o e s o p h a g e a l c a n c e r in m a n y A s i a n c o m m u n i t i e s , including t h o s e w h o h a v e i m m i g r a t e d to t h e U n i t e d K i n g d o m , is b e lieved to b e d u e to n i t r o s a m i n e s f o r m e d a f t e r c h e w i n g b e t e l n u t [ 6 ] w h e t h e r plain o r w r a p p e d into a p i p e r b e t e l l e a f ' q u i d ' . This h a b i t persists as is e v i d e n t f r o m m o u t h staining a n d the w i d e s p r e a d availability of A r e c a nuts in local s h o p s in t h e T o w e r H a m l e t s a r e a of the east e n d of L o n d o n . T h e r e is g o o d e v i d e n c e t h a t c e r t a i n n i t r o s a m i n e s a n d n i t r o s a m i d e s are d i a b e t o g e n i c in a n i m a l s a n d in m a n , including the wellk n o w n d i a b e t o g e n i c a g e n t s t r e p t o z o t o c i n ( S T Z ) a n d the r a t p o i s o n Vacor. T h e s e a g e n t s p r o v o k e T y p e 1 (ins u l i n - d e p e n d e n t ) d i a b e t e s a f t e r single large d o s e s whilst b o t h i n d u c e T y p e 2 d i a b e t e s at l o w e r d o s e s as d o e s S T Z in rats if g i v e n within 2 d a y s of b i r t h [ 7, 8 ]. Specific n i t r o s a m i n e s f r o m I c e l a n d i c s m o k e d m u t t o n , w h i c h m a y b e d i a b e t o g e n i c in m a n , i n d u c e d i a b e t e s in the o f f s p r i n g o f C D 1 m i c e fed b e f o r e m a t i n g [ 9 ]. Since n i t r o s a m i n e s are p r e s e n t in b e t e l n u t a n d also f o r m e d a f t e r i n g e s t i o n ( f r o m A r e c a l alkaloids), the possibility t h a t b e t e l c o n s u m p t i o n m i g h t b e d i a b e t o g e n i c a n d c o n t r i b u t e to t h e i n c r e a s e d p r e v a l e n c e o f T y p e 2 d i a b e t e s f o u n d in A s i a n s w a r r a n t e d investigation. T h i s r e p o r t d e s c r i b e s a s t u d y in the m o u s e as p a r t o f a l a r g e r invest i g a t i o n o f t h e h y p o t h e s i s [ 10 ]. Materials and methods Adult CD1 (random bred) mice as used in earlier studies with Icelandic smoked cured mutton [ 9 ] because of their low spontaneous incidence of diabetes (< 0.05 %) were obtained from Charles River Co. (Manston, Kent, UK). The incidence of diabetes in control animals studied in Aberdeen (Group 1) was 0 % in male and 1.2 % in female mice and in animals studied in London (Group 2) it was 0 %. Only mice confirmed as having a normal intraperitoneal glucose tolerance test (IPGTT) at age 68 weeks were used for feeding experiments and subsequent breeding. The normal feed was Rat and Mouse i (RM1) pellets (SDS, Witham, Essex, UK) low in nitrosamines at under 0.2 gg/kg [ 9 ]. Areca catechu (betel nut) was purchased locally and ground to a powder for incorporation into feed for Group 1 at 30 % in pure corn starch biscuits (Boots Pharmaceuticals, Nottingham, UK) and for Group 2 at 20 % into reconstituted RMI pellets. These feeds were given on alternate days over 4-12 days (Group 1), or pair-fed for 5 consecutive days (Group 2), i.e. fed for 2-6 days, much as in the earlier work with smoked mutton [91. Adult mice fed RM1 pellets throughout the study were used as controls for Group i mice and follow-up glucose tolerance studies were carried out on 2-3 occasions from 10 days to 12 weeks after completion of test feeding. Reconstituted RM1 pellets without betel were pair-fed to control Group 2 mice during test feeding at age 4-6 weeks and glucose tolerance re-assessed on 2-3 occasions at age 12-24 weeks. Eight animals in Group 2 were fed betel nut similarly but with added fl-carotene, known to reduce damage to DNA in human betel chewers [ 11 ], at 15 mg per 100 g of feed. Breeding commenced 14 days after the end of test or pair-test feeding. The glucose tolerance of F1 offspring has been followed from 7-24 weeks, Adults F1 Offspring 90-min plasma glucose (mmol/1) mean + SD (range) n with I G T ap < 0.001;p = NS for all other comparisons of mean 90-min plasma glucose levels in test vs control groups M I M Q l I 1 0/4 2/5 F M 8.2+1.46 12.8:1:2.38 (6.9-10.2) (9.5-16.8) I I 114 115 F M 8.2t:2.38 12.1:1:2.28 (6.0-14.9) (8.4-17.3) I I 0/6 018 F M 8.6+0.91 9.4:1:1.24 (7.3-10.3) (8.0-11.3) and of second and third generation litters for up to 3 months in G r o u p 1 animals. Glucose tolerance has been assessed on two occasions up to 24 weeks of age in F1 offspring of G r o u p 2 animals. Glucose tolerance was assessed by IPGTF, as previously described [ 9 ], using 0.1 ml/g body weight of 20 % glucose in 0.9 % NaC1 i.p. after an 18-h overnight fast (water ad libitum), with blood sampling at 90 rain. Initial studies showed that fasting plasma glucose levels were not significantly raised in glucose intolerant animals, as was found in feeding studies with smoked cured mutton [ 9 ]. All studies therafler used single point 90 min sampling IPGTT's. The plasma glucose levels given are the average of duplicate measurements using a Beckman glucose analyser; coefficient of variation 1.8 % at 4.65 mmol/1, 1.9 % at 9.76 mmol/1 for Group i and 2.1% at 8.22 mmol/1, 2.05 % at ll.8mmol/1 for Group 2; triplicate measurements were m a d e whenever possible and especially for higher levels. Significantly impaired glucose tolerance (IGT) was diagnosed by 90-min blood glucose levels more than 3.0 SD above the m e a n 90-min blood glucose level for the relevant control animals, i.e. at over 16.18 mmol/1 in males and over 10.9 mmol/1 in females in Group 1 (mean normal 90-min levels 8.8 mmol/1 (SD 2.46) in 126 males and 6.16 (SD 1.38) in 86 females), and for G r o u p 2 mice at over 15.8 mmol/1 in males and over 10.6 mmol/1 in females (mean normal 90-min levels 9.8 mmol/1 (SD 2.0) in 40 males and 7.6 mmol/1 (SD 1.0) in 23 females). Histological examination of pancreatic tissue from Group 2 adults and offspring with and without IGT was carried out after standard staining with haematoxylin and eosin of a randomly-selected section through the length of each gland. Planimetry of every islet seen in one such section from each of 84 animals killed aged 6 months in Group 2 (randomly selected and blinded by an independent worker before measurement) was carried out with a Magiscan image analysis system (Applied Imaging, Sunderland, UK) using standard histomorphometric techniques [ 12 ]. Group 1 mice were not examined in significant numbers as they were in use for breeding. Measurements made included the areas of the islets with and without the acinar tissue. The percentage of islet tissue and the numbers of islets per unit area of pancreas were then determined, as well as islet area per gramme body weight. Statistical analysis Statistical analyses included Chi-square tests (corrected for continuity) and Standardized Incidence Ratios (with 95 % confidence limits) for the incidence of IGT and Student's two-tailed t-tests of the differences between mean blood glucose levels [ 13 ]. Results ap <0.01; bp <0.001; : p <0.0001 Showing number of animals examined, mean values and ranges (rain-max) of islet and pancreatic areas, islet/pancreatic area ratios (as %), numbers of islets/pancreas per animal examined and per unit area of pancreas 53 moglycaemic, i of 14 male and 1 of 11 female offspring had IGT; m o r e o v e r the m e a n 90-min plasma glucose levels of male progeny of any G r o u p 1 betel-fed parent with I G T was significantly raised (mean 13.6 mmol/1) compared to those of betel-fed but normoglycaemic parents (mean 8.77 mmol/1,p < 0.01). There was no difference in the corresponding means of female progeny (7.27 and 6.2 mmol/1, respectively). The incidence of I G T in the offspring of control-fed G r o u p 2 animals (Table 2) was nil but was significant (p < 0.005) for male and female offspring of matings of male or female betel-fed animals with control-fed animals. The overall incidence of I G T in F1 offspring of the various betel-fed parents in Groups I and 2 was significant (p < 0.005), 14 of 60 in males and 11 of 55 in females with an SIR of 105.5 (95 % confidence limits 11.2%985.5). There were no progeny that survived to b e w e a n e d from matings between betel-fed adults w h e n b o t h had IGT. The important role of the betel-fed father even when normoglycaemic is shown b y the appearance of I G T in progeny from matings with normal control females, and by the positive relationship found for the combined G r o u p 1 and 2 F1 offspring 90-min glucose levels to paternal (p < 0.01), but not to maternal glycaemia. The incidence of I G T in litters of F2 offspring from betel-fed G r o u p 1 grandparents is shown in Figure 1 which also shows that the level of I G T in F1 females did not appear to influence the incidence of I G T i n the next (F2) generation. Four F3 litters descended from G r o u p 1 betel-fed greatgrandparents were tested (in 2 of the 3 litters I greatgrandmother and one grandfather had I G T ) and 1 of 16 males and 0 of 20 females had developed IGT. The mean 90-min plasma glucose levels were 9.5 (7.15-17.1) and 6.5 (5.2-8.4) in males and females, respectively. Table 3 shows the incidence of I G T i n the various groups studied. Examination of the pancreas of betel-fed G r o u p 2 adult mice (Table 4) revealed hyperplasia of some islets in normoglycaemic animals and in those with IGT, b u t without a significant increase in m e a n areas. No hyperplasia was found in animals fed betel with flcarotene. The m e a n area of the islets was larger than normal (i. e. than in F1 offspring control animals), in all G r o u p 2 F1 offspring with a betel-fed parent. The largest islets were found in those whose mothers had b e e n betel fed and themselves had IGT. The least enlarged islets were the offspring without IGT, one of whose parents had b e e n betel fed; whilst the offspring with I G T whose fathers had b e e n betel fed had islets of intermediate size. There were no differences in islet number/unit pancreatic area or in islet/pancreatic area ratios b e t w e e n these groups of animals. M e a n islet areas per gramme b o d y weight were increased in line with islet areas in betel-fed animals and their offspring. Minor increases in periductal lymphocytes near the islets were seen in glucose-intolerant betel-fed adult mice. M a r k e d lymphocytic infiltration was seen in and around the islets of one G r o u p 1 offspring with I G T though the islets of all other such offspring appeared similar to those of betel-fed adult mice with I G T (Fig. 2). Discussion This study demonstrates that feeding betel nut to young adult mice can induce lasting glucose intolerance. The incidence of I G T was greater, both in adults and their subsequent offspring, than that seen in mice fed Icelandic s m o k e d cured mutton or its nitrosamines [ 9 ], and affected animals were well built and healthy. Diabetogenic nitroso-compounds can induce Type 1 or Type 2 diabetes according to dose [ 7, 8 ]. The I G T produced by betel feeding in the present study appears to be non-insulin-dependent. The increased islet size found in betel-fed mice with I G T and their offspring is similar to the changes found b y O k a m o t o et al. [14] after partial p a n c r e a t e c t o m y or exposure to islet toxins with resultant activation of the R E G gene, and islets seem not to have b e e n lost b u t to have hypertrophied at the expense of acinar tissue, though in the absence of weights or volumes for the whole gland this remains speculative. Individual susceptibility to the diabetogenic agent S T Z appears to d e p e n d on M H C type, (class 1 A, 1 E and 1J) [ 15, 16 ], and similar factors m a y determine betel,nut diabetogenicity. The present study shows that the transmission of significant glucose intolerance to offspring was not a simple effect of maternal hyperglycaemia during gestation since F1 glycaemia related to paternal b u t not maternal glycaemia and I G T developed in s o m e offspring of betel-fed male mice m a t e d with betel-fed normoglycaemic females or with normal control female mice. I G T was found in 11% of the F2 generation derived from betel-fed grandparents w h e n neither parent was glucose intolerant. The mechanism by which Nnitroso c o m p o u n d s such as S T Z cause diabetes has b e e n suggested to be the binding of a glucose ring-like moiety in the molecule to beta-cell glucose receptors, leading to D N A damage [ 14, 17 ]. Betel-nut nitrosamines include similar structures [18]. The induction of I G T in p r o g e n y of nitrosamine-treated animals is unlikely to b e due to placental transfer, since clearance of nitrosamines from tissues takes less than 24 h, though it is slower from the testes [ 9 ]. In addition mating began 2 weeks after completion of test feeding and second F1 litters contained diabetic offspring from G r o u p 2 betel-fed adults, as r e p o r t e d with other nitrosamine-induced diabetes [ 19 ]. The major abnormalities reported in the heads of spermatozoa 30 days after completion of 5 days administration of pan masala (a betel-nut containing snack) to adult mice [ 20 ], and the damage to chromosomes in circulating lymphocytes in man and m o u s e following betel consumption suggests a nuclear mutation should be considered [ 21 ], though the m o r e rapid rates of mitochondrial mutation would provide a mechanism for the m a r k e d changes in disease incidence b e t w e e n generations [ 22 ]. Epidemiological studies of risk markers for diabetes in relation to dietary factors including betel chewing carried out in East L o n d o n are being analysed in relation to glycaemia (B. J. Boucher, N. Mannan, unpublished observations). Simple oral supplements o f / 3 - c a r o t e n e or eating betel nut in piper betel vine-leaf w r a p p e d 'quids' (leaves shown to contain compounds suggestive of cartainoids on preliminary s p e c t r o p h o t o m e t r y ) reduces b o t h D N A damage and the incidence of m o u t h cancer in man [ 11, 18 ]. These observations together with the preliminary finding that islets in animals fed betel nut with fl-carotene were smaller rather than larger, and recent work showing that fl-carotene is the most effective antidote to the mutagenic effect of dilute a q u e o u s extracts of betel nut on mouse and human tissues in culture (D. N. Wheatley, J.M. Stowers, unpublished observations) suggest that strategies could be found to reduce the diabetogenicity of nitrosated compounds relevant to man, as demonstrated in experimental animals [14]. Further feeding studies with added r are therefore in progress. Betel nut has b e e n chewed for hundreds of years, and is currently used by 10 % of the world's population [ 23 ], which makes the suggestion that it could be diabetogenic of potential practical importance. Acknowledgements. The authors thank Dr. J.R.A. Pollock of Pollock & Pool PLC for information and helpful discussion on betel-nut nitrosamines and Dr. C. Mitchell, St. Thomas' Hospital for qualitative analysis of Piper betel vine leaves for ~-carotenoids. We also thank the staff of the Animal House of the Medical School of Aberdeen University and Mr. R. 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B. J. Boucher, S. W. B. Ewen, J. M. Stowers. Betel nut (Areca catechu) consumption and the induction of glucose intolerance in adult CD1 mice and in their F1 and F2 offspring, Diabetologia, 1994, 49-55, DOI: 10.1007/BF00428777