The microbial metabolism of tryptophan in rats fed a diet containing 7.5% saccharin in a two-generation protocol

Fd Chem. Toxic. Vol. 23, No. 4/5, pp. 437-444, 1985

0278-6915/85 $3.00+0.1)0 Pergamon Press Ltd

Printed in Great Britain

THE MICROBIAL METABOLISM OF TRYPTOPHAN IN RATS FED A DIET CONTAINING 7.5% SACCHARIN IN A TWO-GENERATION PROTOCOL J. SIMS* and A. G. RENWtCK Clinical Pharmacology Group, University of Southampton, Medical & Biological Sciences Building, Bassett Crescent East, Southampton SO9 3TU, England

Abstract--Sodium saccharin was fed at 7.5% in the diet to rats in a two-generation protocol. Saccharin-treated animals in both generations showed increased urinary excretion of indican. During lactation, the pups of saccharin-fed dams were exposed to elevated levels of indican via the milk. Establishment of the gut flora in pups at weaning in the presence of saccharin was associated with increased caecal size and caecal protein, decreased caecal tryptophanase activity, and increased urine volume and urinary indican excretion. Pups from dams fed saccharin from birth only, showed more-variable responses during the first weeks of life than pups from dams fed saccharin from before conception, due to variations in tryptophanase activity. The various biochemical and physiological changes were detected soon after the pups were weaned, and were found equally in both males and females. After adjustment for body weight, the changes detected were greatest during the first month after weaning.

Introduction Saccharin increases the incidence of bladder tumours in second-generation male rats fed high dietary levels during two-generation cancer bioassays (Arnold, Moodie, Grice et al. 1980; NAS, 1978; Taylor, Weinberger & Friedman, 1980). The recent two-generation cancer bioassay on saccharin (Food Chemical News, 1983) confirmed these results and also reported that male rats given 5 ~ saccharin in the diet from birth onwards developed an incidence of bladder tumours comparable to that observed for male rats exposed throughout the two-generation protocol. This finding is consistent with the data of Arnold et al. (1980) who reported a small increase in tumour incidence in first-generation male rats when saccharin treatment was initiated at 32 days post-partum. Furthermore, rats exposed to saccharin during the in utero period alone did not develop bladder tumours (Food Chemical News, 1983). Thus, exposure to saccharin in early life, but not in utero, appears to be essential to the expression of the tumorigenic response. There is, however, no satisfactory in vivo mechanism to explain how saccharin, which is nucleophilic and does not undergo detectable metabolism (Sweatman & Renwick, 1979) or bind to the D N A of the target organ (Lutz & Schlatter, 1977), produces tumours of the urinary bladder in male rats. The differences in sensitivity between male and female rats, and between males given saccharin early in life or when mature, cannot be explained by the concentration of saccharin within the target organ (Sweatman & Renwick, 1980 & 1982). Thus, either the bladder of the immature male rat is unusually sensitive to the effects of saccharin, or tumour development is the result of an indirect mechanism that is *Present address: Tate & Lyle, Philip Lyle Memorial Research Laboratory, Whiteknights, Reading. 437

unrelated to the concentration of saccharin in the urinary bladder. The dietary levels of saccharin necessary to elicit a tumorigenic response have been reported to alter various physiological and biochemical processes. The renal clearances of saccharin (Sweatman & Renwick, 1980) and indican (Sims & Renwick, 1983), an endogenous metabolite of tryptophan, were reduced at plasma concentrations of saccharin (200-300 #g/ml) similar to those found in rats fed 5 or 7.5% saccharin in the diet. In addition, saccharin produced a major effect on the catabolism of tryptophan by rat intestinal microflora (Sims & Renwick, 1983). Dietary administration of saccharin (0-10%) produced a dose-related increase in the urinary excretion of indican, the main metabolite of indole, which is formed from tryptophan by the gut microflora. The caecal contents of saccharin-fed rats contained increased amounts of indole, which arose from an accumulation of protein and tryptophan in the caecum rather than an increase in the microbial enzyme tryptophanase which convertes tryptophan to indole. These findings are of interest since protein, tryptophan and indole have been implicated as aetiological factors in experimental bladder carcinogenesis. Ross & Bras (1973) reported that the spontaneous incidence of urinary bladder tumours in male rats was increased markedly by feeding a high-protein diet. Tryptophan has been shown to be a promoter of bladder carcinogenesis using initiators such as F A N F T in rats (Cohen, Arai, Jacobs & Friedell, 1979) and mice (Matsushima, 1977), aminobiphenyl and 2-naphthylamine in dogs (Radomski, Radomski & MacDonald, 1977) and 2-acetylaminofluorene in rats (Dunning, Curtis & Maun, 1950). Coadministration of indole and 2-acetylaminofluorene to rats and hamsters has shown that indole is a co-carcinogen for the bladder and is more potent

438

J. SIMSand A. G. RENWlCK NH= CHz CH - COOH H

Tryptophanaae Gut Flora

H

Indole

Tr]fptophen

Tissues

Absorption

3-Hydroxykynurenine 3-Hydroxyanthranilat e

Hepat i c Oxidation

~

4-

Xanthurenic acid I ndoleacetic acid etc.

OH H

Indoxyl Sulphate " - ~

Hepatic Conjugation

~

OSOzH H

I ndican (Indoxyl Sulphate)

Excr, ,tion Fig. 1. The metabolism of tryptophan to indole and indican.

than the parent amino acid tryptophan (Dunning & Curtis, 1958; Oyasu, Kitajima, Hopp & Sumie, 1972). The increased formation of indole by the gut bacteria and the renal excretion of its main metabolite indican (Fig. 1) in rats fed saccharin-containing diets represents a factor that would contribute to the selection of the urinary bladder as the target organ for saccharin-induced effects. The gastro-intestinal tract of the neonate is sterile at birth, becomes colonized primarily by large populations of lactobaciUi during the first week after birth and then, a few days after weaning, acquires the stable adult flora (Donaldson, 1964; Savage & McAllister, 1971). The adult flora is dependent on species, diet and environmental conditions (Smith, 1965) and thus the effects of saccharin on the microbial catabolism of tryptophan during short-term single-generation feeding studies (Sims & Renwick, 1983) may not apply to a two-generation protocol. The present study was designed to investigate the influence of a two-generation protocol on the changes

in microbial catabolism of tryptophan induced by saccharin administration.

Experimental Chemicals. Saccharin (sodium saccharin dihydrate, lot 1743) was provided by the Sherwin Williams Chemical Co. (Cleveland, Ohio). oL-Tryptophan and its metabolites used as reference compounds for high-pressure liquid chromatography (HPLC) were purchased from Sigma Chemical Co. (Peele, Dorset). All other reagents were of analytical or HPLC grade. Saccharin-containing diets were prepared as described previously (Sims & Renwick, 1983). Animals. Fifteen adult, female Charles River (CD) derived Sprague-Dawley rats (c. 240 g) and 20 adult males (c. 330 g) were purchased from Charles River (Margate, Kent). The animals were housed in groups of five, in cages with wire mesh floors about 3 cm above wood shavings, in a room which contained no other strains of rats and which was maintained at

Tryptophan metabolism: two-generation saccharin study

439

Table 1. Urinary excretion of indican by male rats after 5 wk of eating normal diet, or diet containing 7.5% saccharin, ad lib. Control 7.5% Saccharin Parameter group group Urine vol (ml) 18.9 (6.8) 21.8 (4.6) Indican concn (#g/ml) 142 (63) 298 (70)*** lndican excretion (mg/24hr) 2.35 (0.34) 6.33 (1.22)*** Results are means for ten animals (with the SD in parentheses). Values marked with asterisks are significantly different from the control values (***P <0.001; Student's t test for unpaired data). 22 + 3°C with a light cycle from 07.00 to 21.00 hr. They were given food ( C R M X expanded feed pellets; R H M , Poole, Dorset) and water ad lib. After 2 wk of acclimatization, the animals were weighed and randomly allocated to normal diet (10 males, 10 females) or diet containing 7 . 5 ~ sodium saccharin (10 males, 5 females). Urine samples were collected from the male animals 5 wk after commencing saccharin treatment and analysed for indican by H P L C . One week later the animals were paired for mating; vaginal plugs indicating the start of gestation were detected within a few days in 14 out of the 15 females. The five dams fed diet containing 7 , 5 ~ sodium saccharin were maintained on that regimen and their pups were weaned onto the same diet (Ft group). Five of the females on normal diet were maintained on that diet and their pups were weaned onto normal diet (control group). F o u r of the females on normal diet were fed diet containing 1~o sodium saccharin during the first week post-partum, 3 ~ sodium saccharin during the second week and 7.5~o sodium saccharin from 14 days post-partum onwards; their pups were weaned onto a diet containing 7.5~o sodium saccharin. This last group represent a singlegeneration exposure to saccharin from birth (F0 group) and is comparable to a group fed 5 ~ saccharin in the I R D C study (Food Chemical News, 1983). At 7 and 14 days post-partum, one pup from each litter was selected at random and killed, and the body weight and sex were recorded. Samples of the stomach contents, caecal contents (if available) and urine were collected and analysed for saccharin, indican and other tryptophan metabolites by HPLC. On day 19, the dams were separated from their pups for 2 hr and milk samples were collected by palpation. The dams were then returned immediately to their litters. At weaning (3 wk post-partum), 2-3 males from each litter were placed in metabolism cages for 24-hr urine

collection. Similarly, 2 wk later (5 wk post-partum) 2-3 females from each litter were placed in metabolism cages for 24 hr urine collection. These animals were identified by earmarks and used in the subsequent 24-hr urine collections, which were made at intervals up to 32 wk post-partum. At the time of each urine collection, samples of plasma and caecal contents were taken from an additional three animals of each sex per treatment group. These samples were analysed for saccharin and tryptophan metabolites by H P L C and for caecal tryptophanase activity. The weaned animals were housed in groups of three per cage according to litter and sex. At 32 wk, the animals used for urine collections were killed and samples of plasma and caecal contents taken for analysis. Analytical methods. Methods used for H P L C and analysis of caecal protein content and tryptophanase activity have been described previously (Sims & Renwick, 1983).

Results

Parental generation The excretion of indican was increased significantly in the adult male rats that had been given 7.5~ saccharin in the diet 5 wks prior to mating (Table 1). However, unlike previous studies, the volume of urine was not increased in these rats and thus the concentration of indican was also elevated significantly. The groups of lactating females had similar body weights at weaning but the weight of the caecal contents was increased significantly in both groups receiving saccharin (Table 2). Tryptophanase activity in the caecal contents of control lactating females (0.34gmol/g/hr) was similar to that reported previously in normal males (0.35/~mol/g/hr; Sims & Renwick, 1983). The concentration of tryptophanase

Table 2. Effectsof 7.5% saccharin givenin the diet from before conception (Ft) and 1-7.5% saccharin given in the diet during suckling only (F0) on various parameters in dams at 21 days post-partum Group... Control F~ F0 Parameter No./group... 5 5 4 Body weight (g) 327 (6) 328 (17) 332 (12) Caecal contents (g) 9.0 (2.3) 21.7 (5.0)*** 20.4 (1.7)*** Caecal contents (g/kg) 27.4 (7.3) 65.7 (12.1)*** 61.6 (6.9)*** Caecal tryptophanase (#mol/g contents/hr) 0.34 (0.12) 0.11 (0.06)** 0.00 (0.01)** (#mol/total contents/hr) 2.88 (0.70) 2.51 (1.71) 0.02 (0.03)*** Plasma Saccharin (/~g/ml) -429 (56) 457 (45) Indican (/~g/ml) 2.47(0.74) 20.89(4.02)*** 1.92(1.14) Tryptophan (#g/ml) 18.22(2.98) 12.61 (0.71)** 15.94(3.21) Indolelactic acid (/ag/ml) 0.12 (0.06) 1.10 (0.29)*** 3.33 (0.51)*** Results are means (with the SD in parentheses). Values marked with asterisks are significantly different from the control values (**P < 0.01; ***P < 0.001; Student's t test for unpaired data).

440

J. SlMS and A. G. RENWICK Stomach

contents

I

Milk I

i

i

10, Z E ::L

partum and the absence of tryptophanase 2 days later (Table 2) reflect the increase in dietary concentration of saccharin to 7.5~o.

Second generation 8

~6

c 4 g 2

7 Days

1 4 Days

19 Days

Fig. 2. The concentration o f indJcan in the stomach contents o f rat neonates at 7 and 14 days post-partum and in the milk o f the dams at ]9 days. The results are means (with the SD

given by a vertical line) for five controls (I-q), five F I group rats (1~) and four F 0 group rats (U). See text for explanation of F~ and F 0 group treatments. was reduced significantly in Ft lactating females but due to caecal enlargement the total activity in the caecum was similar. The F0 lactating females, which had received 7.5~ saccharin for only 1 wk at sacrifice, showed a marked loss of this microbial enzyme. The maternal plasma concentrations of indican in the F~ animals, and of indolelactic acid in both the Ft and F0 groups were increased significantly compared with controls (Table 2). The concentration of indican in the maternal milk at 19 days post-partum was elevated markedly in F~ animals compared with controls (Fig. 2). This was reflected in elevated concentrations of indican in the stomach contents of the F~ pups, as measured at 7 and 14 days post-partum (Fig. 2). However, the large differences between individuals made statistical significance marginal. The F0 pups had normal levels of indican in the stomach at 7 days (when the dams were receiving 1~ saccharin diet) but reduced levels at 14 days (when the dams were receiving 3~o saccharin diet). This decrease is consistent with the transient loss of tryptophanase when rats are given high dietary levels of saccharin (Sims & Renwick, 1983). The very low concentrations of indican in the milk of F0 dams at 19 days post-

The body weights of pups were similar in all groups during the first 2 wk post-partum but were depressed significantly in male F t and F0 animals from 3 wk onwards (Table 3). The body weights of females showed a similar trend but did not reach the same statistical significance. At 3 2 w k post-partum the body weights of all saccharin-treated animals were 1 0 - 2 0 ~ lower than controls despite a significant increase in food consumption (Table 4). The volume of urine produced daily by male and female animals of both saccharin-treated groups increased markedly once the animals were weaned onto the solid diet (Table 5). At 32 wk the urine volume in all saccharin-treated animals was two to four times higher than in controls. The weight-adjusted results showed a maximum increase in urine volume between 4 and 7 wk post-partum. The excretion of indican was increased significantly in the Ft males and was two to three times that found in control animals from 3 wk of age (Table 6). After adjustment for body weight the maximum excretion of indican occurred between 3 and 7 wk of age in both control and Ft groups. There were large differences in indican excretion between individual males in the F 0 group, especially soon after weaning, and several animals had negligible amounts of indican in their urine (3 animals at 4 wk, 2 at 7 wk and 2 at 11 wk). Despite these low values the mean excretion (mg/kg/24 hr) was similar to that of the control group by 4 wk. By 7 wk, the indican excretion in male F0 animals was increased compared to controls and was similar to that of the F~ group from 11 wk onwards. The excretion of indican by female offspring showed a similar pattern (Table 7). The maximum excretion of indican (mg/kg/24 hr) occurred at 5 wk and again the F 0 animals showed large variations between individuals in the early parts of the study. At 3 2 w k indican excretion was three to four times greater in saccharintreated animals than in controls. There were no consistent sex differences in indican excretion after correction for body weight. The concentration of tryptophan in plasma

Table 3. Body weights of second-generation rats fed 7.5~ saccharin in the diet, from groups with dams fed 7.5~ saccharin in the diet from before conception (F 0 or 1-7.5~ saccharin in the diet during suckling only (F0) Body weight (g) Males Females Age (wk) Control Ft F0 Control FI F0 It 13.4(0.6) 14.5(1.4) 14.0(1.8) 2t 24.1 (3.3) 24.4(1.6) 25.3 (2.7) 3 41.6 (4.3) 34.6 (3.8)** 33.7 (3.9)*** 4 80 (5) 60 (12)*** 60 (10)*** 94 (7) 72 (15)*** 88 (8) 5 7 235 (14) 193 (24)*** 184 (21)*** 169(12) 156(17) 162(4) 8 11 381 (25) 308 (24)*** 320 (30)*** 255 (21) 229 (22)* 238 (12)* 15 476 (37) 393 (33)*** 410 (31)*** 322 (76) 275 (2 l) 272 (19) 325 567 (36) 476 (40)*** 511 (47)*** tPooled data for approximately three males and three females per group. ~Seven animals per group. Results are means for ten animals (with the SD in parentheses) except where indicated. Values marked with asterisks are significantlydifferent from control values (*P < 0.05; **P < 0.01; ***P < 0.001; Student's t test for unpaired data).

Tryptophan metabolism: two-generation saccharin study

441

Table 4. Food consumption of second-generation rats fed 7.5% saccharin in the diet, from groups with dams fed 7.5Yo saccharin in the diet from before conception (F 0 or 1-7.5% saccharin in the diet during suckling only (F0) Food intake (g/day) Age (wk) 3 4 5 7 8 11 15 32

Control 5.8 (1.2) 12.0(0.6)

Males FI 3.2 (1.1)*** 11.0(4.3)

F0 1.0 (I.2)*** 11.1 (1.5)

19.0 (2.6)

20.0 (3.4)

22.4 (4.7)

13.7 (3.1) 10.9 (4.5) 9,3 (5.8)

22.1 (4.4)** 23.2 (3.9)*** 20.0 (2.8)***

20.1 (4.5)** 20.2 (4.0)*** 21.1 (4.2)***

Females F]

Control

F0

11.9(1.6)

13.0(2.9)

13.0(1.9)

14.3 (1.4)

22.3 (7.5)*

19.7 (3.3)**

11.0 (2.1) 6.0 (5.0)

17.2 (6.5) 24.3 (3.7)***

14.7 (3.3)* 16.9 (5.2)**

Results are means for seven animals (with the SD in parentheses). Values marked with asterisks are significantly different from control values (*P < 0.05; **P < 0.01; ***P < 0.001; Student's t test for unpaired data). Table 5. Volume of urine excreted by second-generation rats fed 7.5% saccharin in the diet, from groups with dams fed 7.5% saccharin in the diet from before conception (FL) or 1-7.5% saccharin in the diet during suckling only (F0) Urine excretion (ml/kg/24 hr) Age (wk) 3 4 5 7 8 11 15 32

Males

Females

Control

Ft

F0

61 (24) 54(17)

61 (19) 127(28)***

49(17) 124(31)***

36 11)

93 (18)***

104(19)***

28(18) 31 ( 1 2 ) 21 (10)

64(19)*** 65(16)*** 61 (13)***

65(19)*** 72(30)*** 81 (32)***

Control

FI

F0

42 (7)

155 (46)***

100 (26)***

46 (8)

120 (45)***

106 (36)***

47(26) 48 (34)

84(47)* 108 (34)**

94(38)** 104 (30)**

Results are means for ten animals (with the SD in parentheses) except for results at 32 wk, when seven animals were used. Values marked with asterisks are significantly different from control values (*P < 0.05; **P < 0.01; ***P < 0.001; Student's t test for unpaired data).

( T a b l e 8) w a s r e d u c e d slightly in s a c c h a r i n - t r e a t e d a n i m a l s , especially s o o n after w e a n i n g . I n c o n t r a s t , the c o n c e n t r a t i o n s o f i n d i c a n a n d indolelactic acid in the p l a s m a were increased significantly in b o t h the F~ a n d F0 g r o u p s . A t 32 w k the p l a s m a c o n c e n t r a t i o n o f i n d i c a n w a s m o r e t h a n five times g r e a t e r in all s a c c h a r i n - t r e a t e d a n i m a l s t h a n in c o n t r o l s . T h e c a e c u m w a s n o t discernible in 1-wk-old p u p s ,

Table 6. lndican excretion by male second-generation rats fed 7.5~ saccharin in the diet, from groups with dams fed 7.5% saccharin in the diet from before conception (F~) or 1-7.5% saccharin in the diet during suckling only (F0) Age (wk)

Indican excretion Control

FI

F0

(rag/24 hr) 3 4 7 11 15 32

0.19 (0.09) 1.03 (0.21) 2.51 (0.68) 2.41 (0.67) 2.94(0.61) 2.50 (0.76)

0.29 (0.10)* 1.89 (0.73)** 4.42 (1.07)*** 6.54 (1.04)*** 7.99(1.63)*** 7.36 (1.67)***

0.08 (0.04)** 0.55 (0.5 I)* 3.05 (2.38) 5.55 (3.07)** 8.95(2.12)*** 5.97 (2.92)*

(mglkg[24 hr) 3 4 7

4.6(1.7) 12.9 (2.4) 10.6 (2.6)

8.2(2.3)*** 31.0 (11.0)*** 23.2 (5.6)***

2.4(1.1)** 9.7 (9.5) 17.9 (14.4)

11

6.4 (1.8)

21.3 (3.8)***

17.9 (9.9)**

15 32

6.2(1.4) 4.5(1.6)

20.5(4.6)*** 15.3(2.3)***

22.1 (6.1)*** 11.4(5.3)**

Results are means for ten animals (with the SD in parentheses) except for results at 32 wk, when seven animals were used. Values marked with asterisks are significantly different from control values (*P < 0.05; **P < 0.01; ***P < 0.001; Student's t test for unpaired data).

b u t by 2 w k a slight t h i c k e n i n g w a s a p p a r e n t at the a p p r o p r i a t e site. By w e a n i n g , the c a e c u m w a s clearly identifiable a n d b o t h the Ft a n d F 0 a n i m a l s s h o w e d a significantly increased w e i g h t o f caecal c o n t e n t s t h r o u g h o u t the rest o f the s t u d y ( T a b l e 9). T h e m a x i m u m caecal e n l a r g e m e n t ( g / k g b o d y w e i g h t ) o c c u r r e d b e t w e e n 4 to 7 wk, w h i c h c o r r e s p o n d s to the period of greatest food intake per kilogram body weight. T h e total a m o u n t s o f t r y p t o p h a n a s e activity in the caecal c o n t e n t s o f s a c c h a r i n - t r e a t e d p u p s ( F o a n d F~) were r e d u c e d significantly at w e a n i n g ( T a b l e 10).

Table 7. Indican excretion by female second-generation rats fed 7.5~o saccharin in the diet, from groups with dams fed 7.5% saccharin in the diet from before conception (Ft) or 1-7.5~ saccharin in the diet during suckling only (F0) Age (wk)

Indican excretion Control

Fi

F0

(rag/24 hr) 5 8 15 32

I. 17 (0.34) 2.04 (0.35) 2.28(0.80) 1.72 (0.54)

3.09 (0.47)*** 4.06 (0.74)*** 5.18(2.01)*** 6.36 (2.19)***

1.31 (0.96) 3.25 (2.36) 5.82(2.28)*** 5.26 (0.99)***

(mg/kg/24 hr) 5 8 15 32

12.4(3.1) 12.1 (2.0) 9.0(3.0) 5.5 (1.6)

44.1 (9.6)*** 26.3 (4.8)*** 23.1 (9.8)*** 23.0 (8.2)***

14.5(9.9) 19.6 (14.0) 24.3 (9.2)*** 19.5 (4.0)***

Results are means for ten animals (with the SD in parentheses) except for results at 32 wk, when seven animals were used. Values marked with asterisks are significantly different from control values (***P < 0.001; Student's t test for unpaired data).

442

J. SIMS a n d A. G . R E r c w l c g

Table 8. Plasma concentrations of saccharin, tryptophan, indican and indolelactic acid in second-generation rats fed 7.5% saccharin in the diet, from groups with dams fed 7.5% saccharin in the diet from before conception (F~) or I-7.5% saccharin in the diet during suckling only (F 0) Plasma concentration ~ g / m l ) Group

Age ( w k ) . . .

3

4

7

15

32 (males)

32 (females)

184(85)

190(111) 218(83)

368(95) 249(50)

0.6(0.7) 3.9(I.2)*** 3.1 (I.0)***

0.7(0.4) 6.2(2.6)*** 5.8 (I.8)***

23 (4) 20(4) 18(3)*

26 (8) 21 (5) 21 (2)

0.05(0.09) 0.2 (0.2)* 0.4 (0.4)*

0.05(0.11) 0.4 (0.5)* 0.2 (0.2)*

Saccharin

F~ F0

112 (30) 95 (38)

197 (89) 191 (51)

218 (49) 248 (38)

Control F~ F0

0.7 (0.6) 3.3 (1.1)*'* 2.0(1.5)

3.0 (I .0) 6.5(3.4)* 7.5(4.1)*

2.4 (I. 1) 9.3 (4.5)'* 5.5(3.5)

-ladlean

1.2(1.1) 8.0 (3.1)*" --

TryptophM Control Fj F0

18 (1) 19 (4) 16 (3)

17 (3) 14 (6) 15 (2)

17 (4) 12 (2)* 13 (2)

17 (3) 16 (5) --

Indolelacti¢ acid

Control F, Fo

0.2 (0. I ) 0.6 ( 0 . 1 ) " " 0.4 (0.2)***

0.2 (0.1) 0.8 ( 0 . 4 ) " 1.7 (0.5)***

0.1 (0.2) 0.6 (0.4)" 0.6 (0.2)*

0. I (0. I ) 0.4 (0.3)"

Results are means for six animals (three of each sex; with the SD in parentheses) except for results at 32 wk, when seven animals per sex were used. Values marked with asterisks are significantly different from control values (*P < 0.05; **P < 0.01; ***P < 0.001; Student's t test for unpaired data).

However, the activity increased such that by 4 wk in F~ pups and 7wk in the F 0 group the levels were comparable to control levels. The levels of tryptophanase activity in the F 0 group showed a wide variability between individuals up to 7wk postpartum. At 32 wk, the total tryptophanase activities were similar in all groups. There was a twofold increase in the total amount of tryptophan in the caecal contents of F~ and F 0 groups at 4 wk (Table 11) which increased to greater than 25-fold at 32 wk. There was no significant sex difference in the amount of tryptophan among the saccharin-treated animals at 32wk. The total amount of indole was increased significantly in all saccharin-treated groups at 32 wk and there were no significant sex differences. Younger treated animals showed a less consistent increase. Indolelactic acid was increased markedly in both F~ and F0 animals from 3 wk onwards, compared with the negligible levels found in control animals (Table 11). By 32 wk, indolelactic acid was increased 30-fold or more in all saccharin-treated animals. Although the levels were Table 9. Weight of caecal contents in second-generation rats fed 7.5% saccharin in the diet, from groups with dams fed 7.5% saccharin in the diet from before conception (Fj) or I-7.5% saccharin in the diet during suckling only (F 0) Age

_.

Caecal contents (g/kg)~

(wk)

Control

F~

Fo

2~. 3 4 7 15 32 (M) 32 (F)

3.3 (0.4) 12.3 (2.0) 18.6(1.5) 16.2 (I.8) 10.9(3.5) 8.2(3.0) 10.3(2.6)

4.8 (1.3)* 20.7 (7.7)* 56.1 (6.7)*** 57.5 (9.7)*** 44.0 (6.0)*** 35.9(6.0)*** 48.9(13.0)***

3.2 (0.9) 19.6 (7.4)* 52.2 (15.9)*'* 60.4(3.7)*** -39.5 ( 9 . 2 ) " * 40.2(8.7)***

M = Male

F = Female

"['No caecum was discernible in l-wk-old pups. ::[:The weights include the wall of the caecum. Results are means for six animals (three of each sex; with the SD in parentheses) except for results at 32 wk, when seven animals of each sex were used. There was no sex difference in results adjusted for body weight. Values marked with asterisks are significantly different from control values (*P < 0.05; ***P <0.001; Student's t test for unpaired data).

higher in males than in females, the difference was not statistically significant after adjustment for body weight. The total amounts of tryptamine were decreased in both saccharin-treated groups and in both sexes from 3wk onwards. The amounts of indolepropionic and indoleacetic acids were low generally, and showed a much less consistent decrease due to saccharin administration (data not shown). However, the F 0 group had very high levels of indoleacetic acid in the caecum at 7 wk compared with controls. This arose from two F 0 animals that did not possess measurable tryptophanase activity and produced large quantities of indoleacetic acid instead of indole. The amounts of total protein and protein nonprecipitable by trichloroacetic acid in the caecal contents were increased significantly in all saccharintreated animals (Table 12). After adjustment for body weight there was no sex difference in these data. Table 10. Tryptophanase in caecal contents of second-generation rats fed 7.5% saccharin in the diet, from groups with dams fed 7.5% saccharin in the diet from before conception (F0 or 1-7.5% saccharin in the diet during suckling only (F0) Age

__.

(wk)

Control

Tryptophanase activity

3 4 7 15 32 (M) 32 (F)

0.59 (0.21) 0.52(0.18) 0.61 (0.08) 0.40(0.11) 0.54 (0.1 I) 0.61 (0.22)

3 4 7 15 32 (M) 32 (F)

0.30 (0.12) 0.69 (0.22) 1.94(0.29) 1.58 (0.81) 2.51 (1.02) 1.93 (0.80)

Fi

.__ F0

O~mol/g ¢aecal eont~lalbr)

0.10(0.07)*** 0.17 (0.14)** 0.26 (0.1M)*** 0.28(0.04)* 0.17(0.09)*** 0.26 (0.08)'"

0.13 (0.05)**" 0.07(0.07)** 0.20 (0.14)*** -0.14 (0.09)*** 0.22 (0.15)'*

0~ tool/total coatents/hr)

M = Male

0.08 (0.05)'* 0.54 (0.46) 2.47(0.61) 4.31 (1.60)** 2.84(1.57) 2.82 (1.29)

0.09 (0.05)** 0.23 (0.27)** 1.83 (I.21) -2.24(I.93) 2.35 (1.30)

F = Female

Results are means for six animals (three of each sex; with the SD in parentheses) except for results at 32 wk, when seven animals of each sex were used. Values marked with asterisks are significantly different from control values (*P < 0.05; **P <0.01; ***P < 0.001; Student's t test for unpaired data).

Tryptophan metabolism: two-generation saccharin study

443

Table 11. Concentrations of saccharin, tryptophan and tryptophan metabolites in the caeeal contents of second-generation rats fed 7.5% saccharin in the diet, from groups with dams fed 7.5% saccharin from before conception (F,) or 1-7.5% saccharin in the diet during suckling only (F0) Concentration (#g/total caecal contents)t Group Age (wk)... 3 4 7 15 32 (males) 32 (females) Tryptophan Control 23 (13) 107 (45) 322 (166) 40 (20) 184 (67) 62 (25) F, 40(15) 201 (49)** 444(100) 299(126)*** 3991 (1074)*** 3507(956)*** F0 55 (27)* 201 (13)*** 487 (413) -4755 (3029)*** 2732 (385)*** Iadole Control 0.3 (0.2) 15 (7) 44 (8) 23 (24) 44 (32) 19 (14) F~ 1.1 (l.l) 10(4) 56(20) 68(35)* 121 (37)** 156(67)*** Fo 0.7 (0.5) 12 (9) 149 (94)* -111 (68)* 93 (34)*** Indolelactic acid

Control Ft F0

0.1 (0.1) 1.5 (1.5) 4.2 (2.1)**

2 (2) 13 (6)** 46 (23)***

5 (6) 33 (10)*** 68 (55)*

Control Ft F0

4 (3) 2 (4) 1 (1)**

18 (6) 2 (I)*** 2 (l)***

44 (19) 6 (5)*** 9 (6)**

2 (1) 49 (8)*** --

1 (1) 50 (44)* 113 (56)***

1 (1) 37 (29)** 33 (17)***

105 (69) 2 (2)** 4 (6)**

56 (27) 5 (4)*** 2 (2)***

Tryptamine

46 (14) 33 (19) -Saccharin

F, F0

15 (14) 10 (4)

47 (9) 37 (19)

292 (85) 293 (130)

327 (124) --

277 (63) 290 (103)

198 (80) 166 (50)

tExcept for saccharin, where the amounts given are mg/total contents. Results are means for six animals (three of each sex; with the SD in parentheses) except for results at 32 wk, when seven animals of each sex were used. Values marked with asterisks are significantly different from control values (*P < 0.05; **P < 0.01; ***P < 0.001; Student's t test for unpaired data). Table 12. Concentration of protein in the caecal contents of second-generation rats fed 7.5% saccharin in the diet for 32 wk, from groups with dams fed 7.5% saccharin in the diet from before conception (F~) or 1-7.5% saccharin during suckling only (F0) Protein concentration Males Units

Control

Ft

Females F0

Control

F,

F0

16 (2)*** 211 (23)*** 774 (127)***

15 (1)*** 161 (38)*** 593 (146)***

Total protein

mg/g mg/total contents mg/total contents/kg

17 (2) 78 (27) 140 (54)

20 (3)* 342 (59)*** 726 (159)***

17 (1) 343 (82)*** 676 (169)***

mg/g mg/total contents mg/total contents/kg

17 (2) 75 (21) 134 (47)

20 (3)* 17 (2) 338 (60)*** 339 (81)*** 716 (161)*** 668 (168)*** TCA = Trichloroacetic acid

9 (2) 31 (18) 92 (42)

Protein not precipitated by TCA

9 (2) 29 (17) 86 (38)

16 (2)*** 207 (26)*** 759 (139)***

15 (1)*** 161 (38)*** 593 (146)***

Results are means for seven animals (with the SD in parentheses). Values marked with asterisks are significantly different from control values (*P < 0.05; ***P < 0.001; Student's t test for unpaired data).

Discussion

T h e d a t a given in this p a p e r d e m o n s t r a t e t h a t e s t a b l i s h i n g the g u t flora at w e a n i n g in the p r e s e n c e o f s a c c h a r i n d i d n o t alter t h e n a t u r e o f t h e b i o c h e m ical a n d p h y s i o l o g i c a l c h a n g e s o b s e r v e d p r e v i o u s l y (Sims & R e n w i c k , 1983). T h u s , a l t e r e d m i c r o b i a l catabolism of tryptophan with increased formation o f i n d o l e a n d e x c r e t i o n o f i n d i c a n will be i n h e r e n t to t w o - g e n e r a t i o n c a r c i n o g e n i c i t y studies with s a c c h a rin. C o m p a r i s o n o f t h e p r e s e n t findings in 32-wk-old a n i m a l s with p u b l i s h e d d a t a f o l l o w i n g s h o r t - t e r m a d m i n i s t r a t i o n to a d u l t rats (Sims & R e n w i c k , 1983) s h o w s t h a t t h e n e o n a t a l e x p o s u r e d i d n o t influence the magnitude of the changes detected. However, several i n t e r e s t i n g o b s e r v a t i o n s were m a d e d u r i n g the p r e s e n t t w o - g e n e r a t i o n f e e d i n g study. T h e F, m o t h e r s , w h i c h h a d b e e n m a i n t a i n e d o n diet c o n t a i n i n g 7 . 5 ~ s a c c h a r i n f o r 5 w k p r i o r to m a t i n g , h a d a c q u i r e d a caecal m i c r o f l o r a t h a t h a d similar levels o f t r y p t o p h a n a s e activity to t h o s e f o u n d

in c o n t r o l s (Table 2). T h e r e was a t e n - f o l d increase in the c o n c e n t r a t i o n o f i n d i c a n in the p l a s m a o f F1 m o t h e r s . This increase was p r o b a b l y d u e to a c o m b i n a t i o n o f i n c r e a s e d f o r m a t i o n o f i n d o l e in the c a e c u m a n d s a t u r a t i o n o f renal e l i m i n a t i o n o f i n d i c a n , since t h e p l a s m a c o n c e n t r a t i o n s o f s a c c h a r i n (400-500 # g / m l ) were well in excess o f t h o s e r e q u i r e d to i n h i b i t i n d i c a n c l e a r a n c e (Sims & R e n w i c k , 1983). T h e high m a t e r n a l p l a s m a levels o f i n d i c a n resulted in i n c r e a s e d e x p o s u r e o f t h e F1 p u p s via the m a t e r n a l milk (Fig. 2), a n d this was reflected in i n c r e a s e d c o n c e n t r a t i o n s in t h e p u p s ' s t o m a c h c o n t e n t s . It is possible t h a t b o t h the F1 a n d F0 p u p s w o u l d be e x p o s e d to i n c r e a s e d a m o u n t s o f o t h e r e n d o g e n o u s s u b s t r a t e s o f m a t e r n a l renal t u b u l a r secretion. T h e e x c r e t i o n o f i n d i c a n i n c r e a s e d in F1 p u p s as s o o n as they c o n s u m e d s a c c h a r i n diet (Tables 6 & 7). This suggests t h a t t h e s a c c h a r i n - a d a p t e d gut flora o f t h e Ft m o t h e r s was p a s s e d o n to their offspring. T h e F 0 m o t h e r s , w h i c h h a d b e e n fed 7 . 5 ~ s a c c h a rin in t h e diet for 1 w k w h e n t h e p u p s were w e a n e d ,

444

J. S[t,tS and A. G. RENWICK

showed a loss of tryptophanase activity from the caecum (Table 2). A similar initial loss of tryptophanase activity was found when adult males were transferred to a 7.5~o saccharin diet (Sims & Renwick, 1983). This was followed by a variable recovery of activity after 2 to 3 wk of continuous saccharin treatment. This loss of tryptophanase activity in the F0 dams resulted in low concentrations of indican in the maternal milk and plasma (Fig. 2 and Table 2). However, these animals had very high plasma concentrations of indolelactic acid, suggesting that the saccharin-induced increase in this microbial metabolite precedes the increased formation of indole and indican. This loss of maternal tryptophanase was transferred to the F 0 offspring which showed reduced excretion of indican for the first few weeks on 7.59/o saccharin diet (Table 6). This was followed by an increase during the next 8 wk. The pups from two of the four dams showed extremely variable results: some animals used for 24-hr urine collection showed no indican excretion whilst others had no tryptophanase activity in the caecum. These abnormal results affected both males and females and thus were not confined to a single cage of offspring. Pups from the other two F0 mothers gave results comparable to the F t group. An important observation made in the present study was that after adjustment for body weight the maximum increases in most parameters occurred between 3 and 8 w k of age. The results of the two-generation cancer bioassays suggests that feeding saccharin during this time is important in the subsequent development of bladder tumours. There were no statistically significant sex differences apparent in any of the parameters studied that might correlate with the greater sensitivity of the male to tumour development following saccharin administration. The formation of indolelactic acid in the caecum was slightly higher in males than females; this, combined with the very marked increase in the amount present in the caecum of saccharin-treated rats, and the similarity in amounts in F~ and F0 animals in the early weeks of the study, suggest that this bacterial metabolite is worthy of further toxicological investigation. In conclusion, the administration of saccharin to rats in a two-generation protocol results in an increased formation of indole which is a co-carcinogen for the rat urinary bladder. This would contribute to the development of tumours of the urinary bladder under such conditions. However, the absence of marked sex-specific differences in the nature or magnitude of the various effects measured suggests that other factors of equal or greater importance remain to be elucidated. Acknowledgements--The authors are grateful to Mr C. E.

Bunce and Mrs J. A. Joyce for their assistance and expertise in the management of the test animals. The work was funded by a research grant from the Calorie Control Council, Atlanta, Georgia.

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