- Email: [email protected]
Steroids and cancer: Faecal bile acid screening for early detection of cancer risk
STEROIDS AND CANCER: FAECAL BILE ACID SCREENING FOR EARLY DETECTION OF CANCER RISK
PHLS, Centre for Applied Microbiology and Research, Bacterial Metabolism Porton Down. Salisbury. Wiltshire SP4 OJG, England
Research Laboratory,
Summary-The analyses of Beea bife acids in colorecta’t cancer patients, breast cancer patients and he&thy control subjects is described. Faecal excretion of totat bile acids was similar in the three groups, The major bile acids detected were Iithochoiic acid (LCA) and deoxycholic acid (DCA) and the proportions of these (LCA:DCA ratio) were djametr~caily opposed in the colorectal cancer patients (I .91 rk:0.33) and control subjects (5.90 2 0.09). Patients with adenoearcinoma of the breast also exhibited a higher LCA:DCA ratio (1.24 & 0.10) than the control group. The faecal LCA:DCA ratio is an impartant marker of cancer risk especially cancer of the large bowel and it is suggested that it may be a useful adjunct to future screening procedures.
Table 1.tZm.%xmtrtti studies showing a positive correlation between faecal bile acid excretion and eolorectaf cancer Epidemiological evidence suggests that the occurrence of colorectal cancer (CRC) [I, 21 and breast cancer (BC) [3,4] is closely related to dietary intake especially the consumption of animal fat. Whilst the effects of diet on faecai bile acid (FBA) have been finked with carcinagenesis of the large bowel the association of FBA with BC is rather tenuous. The early evidence based on measurement of total FBA excretion has been extremely equivocal. Several studies have shown that FBA excretion is higher in CRC patients (Table 1) compared to controls whilst others have failed ta confirm this (Table 2). Although less documented a similar situation exists for BC (Table 3). Evidence
to support the ~n~~~ye~~~t
- __-Study Hill et al.[s] Reddy and Wynder[6]
Total FBA
6.24 c141* 19.87it 0.94 f31)*
C0ntr& 4.411 (41)* IO.XSIt 0.76 (34If
Results expressed in m&g dry faeces + SEM. *Represents number of subjects in each study. FBA-faecaf biie acids: CRC-wlorectal cancer.
Table 2. Case-control studies showing a negative. correlation between faecal bile acid excretian and colorectal cancer
Studv
Total FBA (mg[g dry faeces) -I_________ CRC. _____ .. . . CcJntrols
of bife acids
in large bowel carcinogenesis has come from animaf model and in citro mutagenicity testing systems (Table 4) and the secondary bile acids lithocholic acid (LCA) and deoxycholic acid (DCA) appear to be particularly important in this respect. Models of BC carcinogenesis have yet to be described. Prior to 1983 all studies pertaining to FBA. CRC and EC utilized the methods of Grundy et al.[t 71 and Evrard and Janssen[tgf. These methods involve strong hydrolytic steps in the work-up procedures, e.g. boiling in strong acid or alkali solutions which cause appreciable degradation and modification of steroid molecules and thus only allowing rather crude measurements of the total or principal bile acids in faeces. *To whom correspondence
._ CRC
Mudd et al. I@ Murray er a,.[91
to.@ -0.90 (19)* 4.53 f 0.27 (37)*
10.7&C I .04 (19Y 7.87 * 0.84 (36)*
Results shown in mg/gdry faeces k SEM. *Indicates the number of subjects studied. FBA-faecal bile acids; CRC-colnrectai cancer.
Table 3. Case-control studies showing an equivocal correlation between faecal bile acid excretion and breast cancer
-_--Study Murray et ol.[101
Papatestas et nf.[lI]
Total FBA (q/g dry faxes)
BC
Controls
5.99 * 0.69 (3OY 17 * 132
7.87 + 0.37’ (36)’ II +@ 159)’
(5QI*
and reprint requesis should be
SW%
391
Results shown in m&g dry Faeces + ‘SEM ‘SD.
*Indicates the number af sub&is studied. FM-faecai bile acid; BC-breast cancer.
RCXIERT W. OWEN et al.
392
Table 4. Evidence for bile acids havinn lumour initiatiw or womotina wooerties Test svstem
Studv Narisawa tf crt.[lZ]
Rectal mstdlation
Silverman and Andrews[l3]
Salmonella mutagenesis
Cohen rr a/.[141 Martin el a/.[151 Wilpart el a/.[l6]
Oral administration Oral admmistration Salmonella mutagenesis
LCA-lithocholic
acid: DCA-deoxycholic
Genotoxic effect
DCA LCA DCA LCA CA CDCA DCA LCA
Co-carcinogenic
aad: CDCA-chenodeoxycholic
Recently more refined methods based on the pioneering efforts of Alme et a/.[191have been developed for the detailed analyses of bile acids. However the modified methods of Tanida et a!.[201 and Setchell et d.[21] whilst using anion-exchange chromatography for the fractionation of faecal extracts are still based on rather crude extraction procedures. Nevertheless this has allowed in-depth studies of the qualitative and quanitative FBA excretion in Japanese subjects (healthy Japanese [22], Japanese with adenomotous polyps [20] and Japanese with CRC [23]). These studies again do not show a positive correlation between FBA (individual and total) and CRC. However it is possible that the aetiology of CRC in Japan with a low incidence rate of 31.3 x 10’ population is different from that in western countries. We have developed a rather more sophisticated method (Owen et a1.[24]) utilizing sequential solvent stripping in a Soxhlet apparatus allied to anionexchange chromatography and have applied this technique for the detailed assay of faecal steroids in different cancer groups. ORJECTIVES
Bile acid
Co-carcinogenic Co-carcinogenic Co-mulagenic
acid; CA--cholic
Faecal samples were obtained and stored at -40’ C until ready for use. After freeze-drying, the samples were ground to a fine homogenous powder. Faecal extracts were obtained by two-stage Soxhlet extraction of dry faeces (5OOmg) using petroleum ether (40-60‘) and chIoroform/methanol (1 :I) to remove sequentially neutral and acidic steroids respectively. The acidic extracts were fractionated by anion-exchange column-chromatography on diethylaminohydroxypropyl-Sephadex into neutral steroids, free bile acids, glycine conjugated, taurine conjugated and sulphated steroids (Owen et al., 1984)[24]. The results of the bile acid assays are reported here. After fractionation the bile acids were quantified by GLC on a Pye 304 gas chromatograph fitted with a 3% OV-I column. The biie acids were separated using a temperature gradient from 16@26O”C at 4”C/min. The bile acids were methylated with ethereal diazomethane and methyl hyodeoxycholate was incorporated as internal standard. RESULTS
This study was designed to compare the faecal bile acid profiles of CRC, BC and healthy subjects. GROUPS
The study groups comprised 34 CRC patients, 16 BC patients and 36 healthy British volunteers. The CRC patients had tumours classified as Dukes A, B or C 1251.Patients with liver involvement (Dukes C,) and intestinal obstruction were excluded from the study. The BC cases all had histologically proven adenocarcinomas.
On examination of the quantitative excretion of FBA (Table 5) there were no significant differences between the study groups. Of the principal bile acids lithocholic acid (LCA) and deoxycholic acid (DCA) but not chenodeoxychohc acid and cholic acid could be detected, The excretion of LCA was significantly higher in the CRC group compared to the control group whilst DCA excretion was comparable in the three groups. By examining the ratios of the secondary bile acids i.e. the ratio of LCA:DCA significant differences could be discerned between both the CRC and BC
Table 5. Composition of major faecal bile acids in the study groups Major FBA’ Grow Colorectal cancer [34] Breast cancer 1161 Healthy contrbls’[36]
acid
METHODS
OF THE STUDY
STUDY
Co-mutagenic
LCA
DCA
CDCA
4.01 + 0.63 3.47 f0.52 2.78 zO.36
3.39 f 0.59 2.96+0.39 3.93 TO.54
-
CA -
Total FBA* 7.40* I.12 6.45 + 0.84 6.71 ? 0.86
*Results expressed in mg/g of dry faeces + SEM; LCA-lithocholic acid; DCAdeoxycholic acid: CDCA-chenodeoxycholic acid; CA--cholic acid; FBA-faecal bile acids.
Steroids and cancer
Fig. I. The LCA:DCA ratio within the study groups. C--controls; BC-breast cancer; CRC-colorectal cancer; LCA-hthocholic acid; DCA-deoxycholic acid.
to the control group (Table 6). The LCA:DCA ratio of the CRC group being double that of the control group (Fig. 1). Another interesting feature has emerged from this study which relates the LCA:DCA ratio to the excretion of total FBA, this being the LCA/ DCA x FBA index (Table 6) which shows a positive correlation with both CRC and BC. groups compared
DISCUSSION
Our findings using more sophisticated analytical techniques are at variance with several recent studies [22,23] but support our original observations in a CRC case-control study (Owen ei a1.[26]). This may be explained in part by the difference in geographical locations of the subjects studied and corresponding prevalence rates of CRC. The present study again highlights that whilst assay of total FBA may be useful for discriminating between low-risk and high-risk CRC populations it has obvious limitations when applied to both CRC and BC case-control studies. The LCA: DCA ratio has again been re-emphasised as a useful marker of CRC risk and together with the LCAiDCA x FBA index shows a positive correlation with both CRC and BC. Of these two new markers the LCA:DCA ratio appears to be a more powerful discriminant because 75% of the CRC group and
Table 6. LCA:DCA
Ratio and LCA:DCA groups
Group
LCA:DCA
Colorectal cancer [34] Breast cancer f 161 Healthy controls 1361 LCA-lithocholic acid.
x FBA index in the study
1.91 f 0.33 1.24+0.10 0.90 + 0.09
acid; DCAdeoxycholic
LCA:DCA
69% of the BC group exhibited a ratio > 1.0 whilst 75% of the control group exhibited a ratio of < 1.0. One would accordingly predict that any person excreting more LCA than DCA must be regarded as a high-risk CRC individual and in the case of women to be at risk also of adenocarcinoma of the breast. Having established that the LCA:DCA ratio is an excellent marker of both CRC and BC risk it stifl remains imperative to discover the reasons for this. Whilst 75% of the CRC group and 69% of the BC group excrete more LCA than DCA it does not necessarily mean that LCA is a more potent promotor of cancer than DCA. For instance Wilpart et 4161 have shown that LCA and DCA are equally potent co-mutagens (in conjunction with 1,2~imethylhydr~ine) in the Ames Salmonella mutagenicity assay, whilst Summerton et uf.[27] have shown that CRC patients carry substantially more DCA receptors in tumour tissue compared to the intestinal mucosa of controls. This indicates that both LCA and DCA may be promoters of CRC. Although LCA has been detected in both breast turnout and normal tissue [28] to date there is no evidence for bile acid receptors being present. Clearly then the role of bile acids as promoters of CRC and BC is not a simple one. However we wish to propose that LCA and DCA are important in the promotion of human cancer and that future studies should concentrate upon screening populations for the new markers of CRC and BC risk, the LCA:DCA ratio and the LCA:DCA x FBA index so that the goal of cancer prevention can be achieved. Acknowiedge~en~~-We are extremely grateful to the Cancer Research Campaign for continued financial support. REFERENCES
1. Haenszel W., Berg J. W.. Segi M., Kurihara
2. 3.
4. 5.
6.
7.
x FBA
1I .23 f
2.30 8.08 + I .46 5.15+0.67
acid; FBA-faecal
8. bile
393
M. and Locke F. B.: Large bowel cancer in Hawaiian Japanese. J. natn. Cancer Inst. 10 (1973) 176551799. Wynder E. L. and Reddy B. S.: Diet and cancer of the colon. Curr Concepts Nutr. 6 (1977) 55-71. Wynder E. L.: Current concepts of the aetiology of breast cancer. In Prognostic Factors in Breast Cancer (Edited by Forrest and Kunkler). Longman, London (1968) p. 32. Drasar B. S. and Irving D.: Environmental factors and cancer of the colon and breast. Br. J. Cancer 27 (1973) 167-172. Hill M. J., Drasar B. S., Williams R. E. 0.. Meade T. W., Cox A. G., Simpson J. E. P. and Morson B. C.: Faecal bile acids and Clostridia in patients with cancer of the large bowel. Lancet 1 (1975) 535-539. Reddy B. S. and Wynder E. L.: Metabolic epidemioiogy of colon cancer: Faecal bile acids and neutral sterols in colon cancer patients and patients with adenomatous polyps. Cancer 39 (1977) 2533-2539. Moskowitz M., White C., Barnett R. N., Stevens S., Russel E., Vargo D. and Floch M. H.: Diet, Faecal bile acids and neutral sterols in carcinoma of the colon. Dig. Dis. Sci. 24 (1979) 746-751. Mudd D. G., McKelvey S. T. D., Norwood W., Elmore D. T. and Roy A. D.: Faecal bile acid concentrations of patients with carcinoma or increased risk of carcinoma in the large bowel. Gut 21 (1980) 587-590.
394
ROBERT W. OWENet al.
9. Murray W. R., Blackwood A., Trotter J. M., Calman K. C. and Mackay C.: Faecal bile acids and Clostridia in the aetiology of colorectal cancer. Br. J. Cancer 41 (1980) 923-928. 10. Murray W. R., Blackwood A., Calman K. C. and Mackay C.: Faecal bile acids and Clostridia in patients with breast cancer. Br. .I. Cancer 42 (1980) 856860. 1 I. Papatestas A. E., Panvelliwalla D., Tartter P. I., Miller S., Pertsemlidis D. and Aufses A. H.: Faecal steroid metabolites and breast cancer risk. Cancer 49 (1982) 1201-1205. 12. Narisawa T., Magadia N. E., Weisburger J. H. and Wynder E. L.: Promoting effect of bile acids on colon carcinogenesis after intrarectal instillation of N-methylN’-nitrosoquanidine in rats. J. natn. Cancer Inst. 53 (1974) 1093-1097. 13. Silverman S. J. and Andrews A. W.: Bile acids; comutagenic activity in the salmonella-mammalianmicrosome mutagenicity test. J. nafn. Cancer Inst. 59 (1977) 1557-1559. 14. Cohen B. I., Raicht R. F., Deschner E. E., Takahashi M., Sarval A. M. and Fazzini E.: Effect of cholic acid feeding on N-methyl-N-nitrosourea induced colon tumours and cell kinetics in rats. J. natn. Cancer Inst. 64 (1980) 573-578. 15. Martin M. S., Justrabo E.. Jeannin J. F., Leclerc A. and Martin F.: Effect of dietary chenodeoxycholic acid on intestinal carcinogenesis induced by 1.2-dimethyl hydrazine in mice. Br. J. Cancer 43 (1981) 884-886. 16. Wilpart M., Mainguet P., Maskens A. and Roberfroid M.: Mutagenicity of 1,Zdimethyl hydrazine towards Salmonella typhimurium; co-mutagenic effect of secondary bile acids. Carcinogenesis 4 (1983) 45-48. 17. Grundy S. M., Ahrens E. H. and Miettinen T. A.: Quantitative isolation and gas-liquid chromatographic analysis of total faecal bile acids. J. Lipid Res. 6 (1965) 397410. 18. Evrard E. and Janssen S.: Gas liquid chromatographic determination of faecal bile acids. J. Lipid Res. 9 (1968) 226-236. 19. Alme B. A., Bremmelgaard A., Sjovall J. and Thomassen P.: Analysis of metabolic profiles of bile acids in
20.
21.
22.
23.
24.
25. 26.
27.
28.
urine using lipophilic anion exchanger and computerized gas-liquid chromatography-mass spectrometry. J. Lipid Res. 18 (1977) 339-362. Tanida N.. Hikasa Y.. Hosomi M., Satomi M., Oohama I. and Shimoyama T.: Faecal bile acid analysis in healthy Japanese subjects using a lipophihc anion exchanger, capillary column gas chromatography and mass spectrometry. Gastroent. Japonica 16 ( 198 1) 363-37 1. Setchell K. D. R.. Lawson A. M.. Tanida M. and Sjovall J.: General methods for the analysis of metabolic profiles of bile acids and related compounds in faeces. J. Lipid Res. 24, (1983) 1085-l 100. Tanida N.. Hikasa Y., Shimoyama T. and Setchell K. D. R.: Comparison of faecal bile acid profiles between patients with adenomatous polyps of the large bowel and healthy subjects in Japan. Gut 25 (1984) 824832. Hikasa Y., Tanida M., Ohno T. and Shimoyama: Faecal bile acid profiles in patients with large bowel cancer in Jaoan. GUI 25 (1984) 833 -838. Owen R. W.: Thompson M. H. and Hill M. J.: Analysis of metabolic profiles of steroids in faeces of healthy subjects undergoing chenodeoxycholic acid treatment by liquid-gel chromatography and gas-liquid chromatography-mass spectrometry. J. steroid Biochem. 21 (1984) 593-600. Dukes C. E.: The classification of cancer of the rectum. J. Pathol. Bact. 35 (1932) 323-332. Owen R. W., Dodo M., Thompson M. H. and Hill M. J.: The faecal ratio of lithocholic acid to deoxycholic acid may be an important aetiological factor in colorectal cancer. Biochem. Sot. Trans. 12 (1984) 861. Summerton J., Flynn M.. Cooke T. and Taylor 1.: Bile acid receptors in colorectal cancer. Br. J. Surg. 70 (1983) 549955 1. Turjman N. and Jacob C.: Distribution of tissue-bound lithocholic acid in normal and neoplastic mammary tissues: does lipid-rich lymph contribute to the transport of lithocholic acid (Abstract). Proceedings of the VI international Bile Acid Meeting (Bile Acids and Lipids) (1980) p. 119.
PHLS, Centre for Applied Microbiology and Research, Bacterial Metabolism Porton Down. Salisbury. Wiltshire SP4 OJG, England
Research Laboratory,
Summary-The analyses of Beea bife acids in colorecta’t cancer patients, breast cancer patients and he&thy control subjects is described. Faecal excretion of totat bile acids was similar in the three groups, The major bile acids detected were Iithochoiic acid (LCA) and deoxycholic acid (DCA) and the proportions of these (LCA:DCA ratio) were djametr~caily opposed in the colorectal cancer patients (I .91 rk:0.33) and control subjects (5.90 2 0.09). Patients with adenoearcinoma of the breast also exhibited a higher LCA:DCA ratio (1.24 & 0.10) than the control group. The faecal LCA:DCA ratio is an impartant marker of cancer risk especially cancer of the large bowel and it is suggested that it may be a useful adjunct to future screening procedures.
Table 1.tZm.%xmtrtti studies showing a positive correlation between faecal bile acid excretion and eolorectaf cancer Epidemiological evidence suggests that the occurrence of colorectal cancer (CRC) [I, 21 and breast cancer (BC) [3,4] is closely related to dietary intake especially the consumption of animal fat. Whilst the effects of diet on faecai bile acid (FBA) have been finked with carcinagenesis of the large bowel the association of FBA with BC is rather tenuous. The early evidence based on measurement of total FBA excretion has been extremely equivocal. Several studies have shown that FBA excretion is higher in CRC patients (Table 1) compared to controls whilst others have failed ta confirm this (Table 2). Although less documented a similar situation exists for BC (Table 3). Evidence
to support the ~n~~~ye~~~t
- __-Study Hill et al.[s] Reddy and Wynder[6]
Total FBA
6.24 c141* 19.87it 0.94 f31)*
C0ntr& 4.411 (41)* IO.XSIt 0.76 (34If
Results expressed in m&g dry faeces + SEM. *Represents number of subjects in each study. FBA-faecaf biie acids: CRC-wlorectal cancer.
Table 2. Case-control studies showing a negative. correlation between faecal bile acid excretian and colorectal cancer
Studv
Total FBA (mg[g dry faeces) -I_________ CRC. _____ .. . . CcJntrols
of bife acids
in large bowel carcinogenesis has come from animaf model and in citro mutagenicity testing systems (Table 4) and the secondary bile acids lithocholic acid (LCA) and deoxycholic acid (DCA) appear to be particularly important in this respect. Models of BC carcinogenesis have yet to be described. Prior to 1983 all studies pertaining to FBA. CRC and EC utilized the methods of Grundy et al.[t 71 and Evrard and Janssen[tgf. These methods involve strong hydrolytic steps in the work-up procedures, e.g. boiling in strong acid or alkali solutions which cause appreciable degradation and modification of steroid molecules and thus only allowing rather crude measurements of the total or principal bile acids in faeces. *To whom correspondence
._ CRC
Mudd et al. I@ Murray er a,.[91
to.@ -0.90 (19)* 4.53 f 0.27 (37)*
10.7&C I .04 (19Y 7.87 * 0.84 (36)*
Results shown in mg/gdry faeces k SEM. *Indicates the number of subjects studied. FBA-faecal bile acids; CRC-colnrectai cancer.
Table 3. Case-control studies showing an equivocal correlation between faecal bile acid excretion and breast cancer
-_--Study Murray et ol.[101
Papatestas et nf.[lI]
Total FBA (q/g dry faxes)
BC
Controls
5.99 * 0.69 (3OY 17 * 132
7.87 + 0.37’ (36)’ II +@ 159)’
(5QI*
and reprint requesis should be
SW%
391
Results shown in m&g dry Faeces + ‘SEM ‘SD.
*Indicates the number af sub&is studied. FM-faecai bile acid; BC-breast cancer.
RCXIERT W. OWEN et al.
392
Table 4. Evidence for bile acids havinn lumour initiatiw or womotina wooerties Test svstem
Studv Narisawa tf crt.[lZ]
Rectal mstdlation
Silverman and Andrews[l3]
Salmonella mutagenesis
Cohen rr a/.[141 Martin el a/.[151 Wilpart el a/.[l6]
Oral administration Oral admmistration Salmonella mutagenesis
LCA-lithocholic
acid: DCA-deoxycholic
Genotoxic effect
DCA LCA DCA LCA CA CDCA DCA LCA
Co-carcinogenic
aad: CDCA-chenodeoxycholic
Recently more refined methods based on the pioneering efforts of Alme et a/.[191have been developed for the detailed analyses of bile acids. However the modified methods of Tanida et a!.[201 and Setchell et d.[21] whilst using anion-exchange chromatography for the fractionation of faecal extracts are still based on rather crude extraction procedures. Nevertheless this has allowed in-depth studies of the qualitative and quanitative FBA excretion in Japanese subjects (healthy Japanese [22], Japanese with adenomotous polyps [20] and Japanese with CRC [23]). These studies again do not show a positive correlation between FBA (individual and total) and CRC. However it is possible that the aetiology of CRC in Japan with a low incidence rate of 31.3 x 10’ population is different from that in western countries. We have developed a rather more sophisticated method (Owen et a1.[24]) utilizing sequential solvent stripping in a Soxhlet apparatus allied to anionexchange chromatography and have applied this technique for the detailed assay of faecal steroids in different cancer groups. ORJECTIVES
Bile acid
Co-carcinogenic Co-carcinogenic Co-mulagenic
acid; CA--cholic
Faecal samples were obtained and stored at -40’ C until ready for use. After freeze-drying, the samples were ground to a fine homogenous powder. Faecal extracts were obtained by two-stage Soxhlet extraction of dry faeces (5OOmg) using petroleum ether (40-60‘) and chIoroform/methanol (1 :I) to remove sequentially neutral and acidic steroids respectively. The acidic extracts were fractionated by anion-exchange column-chromatography on diethylaminohydroxypropyl-Sephadex into neutral steroids, free bile acids, glycine conjugated, taurine conjugated and sulphated steroids (Owen et al., 1984)[24]. The results of the bile acid assays are reported here. After fractionation the bile acids were quantified by GLC on a Pye 304 gas chromatograph fitted with a 3% OV-I column. The biie acids were separated using a temperature gradient from 16@26O”C at 4”C/min. The bile acids were methylated with ethereal diazomethane and methyl hyodeoxycholate was incorporated as internal standard. RESULTS
This study was designed to compare the faecal bile acid profiles of CRC, BC and healthy subjects. GROUPS
The study groups comprised 34 CRC patients, 16 BC patients and 36 healthy British volunteers. The CRC patients had tumours classified as Dukes A, B or C 1251.Patients with liver involvement (Dukes C,) and intestinal obstruction were excluded from the study. The BC cases all had histologically proven adenocarcinomas.
On examination of the quantitative excretion of FBA (Table 5) there were no significant differences between the study groups. Of the principal bile acids lithocholic acid (LCA) and deoxycholic acid (DCA) but not chenodeoxychohc acid and cholic acid could be detected, The excretion of LCA was significantly higher in the CRC group compared to the control group whilst DCA excretion was comparable in the three groups. By examining the ratios of the secondary bile acids i.e. the ratio of LCA:DCA significant differences could be discerned between both the CRC and BC
Table 5. Composition of major faecal bile acids in the study groups Major FBA’ Grow Colorectal cancer [34] Breast cancer 1161 Healthy contrbls’[36]
acid
METHODS
OF THE STUDY
STUDY
Co-mutagenic
LCA
DCA
CDCA
4.01 + 0.63 3.47 f0.52 2.78 zO.36
3.39 f 0.59 2.96+0.39 3.93 TO.54
-
CA -
Total FBA* 7.40* I.12 6.45 + 0.84 6.71 ? 0.86
*Results expressed in mg/g of dry faeces + SEM; LCA-lithocholic acid; DCAdeoxycholic acid: CDCA-chenodeoxycholic acid; CA--cholic acid; FBA-faecal bile acids.
Steroids and cancer
Fig. I. The LCA:DCA ratio within the study groups. C--controls; BC-breast cancer; CRC-colorectal cancer; LCA-hthocholic acid; DCA-deoxycholic acid.
to the control group (Table 6). The LCA:DCA ratio of the CRC group being double that of the control group (Fig. 1). Another interesting feature has emerged from this study which relates the LCA:DCA ratio to the excretion of total FBA, this being the LCA/ DCA x FBA index (Table 6) which shows a positive correlation with both CRC and BC. groups compared
DISCUSSION
Our findings using more sophisticated analytical techniques are at variance with several recent studies [22,23] but support our original observations in a CRC case-control study (Owen ei a1.[26]). This may be explained in part by the difference in geographical locations of the subjects studied and corresponding prevalence rates of CRC. The present study again highlights that whilst assay of total FBA may be useful for discriminating between low-risk and high-risk CRC populations it has obvious limitations when applied to both CRC and BC case-control studies. The LCA: DCA ratio has again been re-emphasised as a useful marker of CRC risk and together with the LCAiDCA x FBA index shows a positive correlation with both CRC and BC. Of these two new markers the LCA:DCA ratio appears to be a more powerful discriminant because 75% of the CRC group and
Table 6. LCA:DCA
Ratio and LCA:DCA groups
Group
LCA:DCA
Colorectal cancer [34] Breast cancer f 161 Healthy controls 1361 LCA-lithocholic acid.
x FBA index in the study
1.91 f 0.33 1.24+0.10 0.90 + 0.09
acid; DCAdeoxycholic
LCA:DCA
69% of the BC group exhibited a ratio > 1.0 whilst 75% of the control group exhibited a ratio of < 1.0. One would accordingly predict that any person excreting more LCA than DCA must be regarded as a high-risk CRC individual and in the case of women to be at risk also of adenocarcinoma of the breast. Having established that the LCA:DCA ratio is an excellent marker of both CRC and BC risk it stifl remains imperative to discover the reasons for this. Whilst 75% of the CRC group and 69% of the BC group excrete more LCA than DCA it does not necessarily mean that LCA is a more potent promotor of cancer than DCA. For instance Wilpart et 4161 have shown that LCA and DCA are equally potent co-mutagens (in conjunction with 1,2~imethylhydr~ine) in the Ames Salmonella mutagenicity assay, whilst Summerton et uf.[27] have shown that CRC patients carry substantially more DCA receptors in tumour tissue compared to the intestinal mucosa of controls. This indicates that both LCA and DCA may be promoters of CRC. Although LCA has been detected in both breast turnout and normal tissue [28] to date there is no evidence for bile acid receptors being present. Clearly then the role of bile acids as promoters of CRC and BC is not a simple one. However we wish to propose that LCA and DCA are important in the promotion of human cancer and that future studies should concentrate upon screening populations for the new markers of CRC and BC risk, the LCA:DCA ratio and the LCA:DCA x FBA index so that the goal of cancer prevention can be achieved. Acknowiedge~en~~-We are extremely grateful to the Cancer Research Campaign for continued financial support. REFERENCES
1. Haenszel W., Berg J. W.. Segi M., Kurihara
2. 3.
4. 5.
6.
7.
x FBA
1I .23 f
2.30 8.08 + I .46 5.15+0.67
acid; FBA-faecal
8. bile
393
M. and Locke F. B.: Large bowel cancer in Hawaiian Japanese. J. natn. Cancer Inst. 10 (1973) 176551799. Wynder E. L. and Reddy B. S.: Diet and cancer of the colon. Curr Concepts Nutr. 6 (1977) 55-71. Wynder E. L.: Current concepts of the aetiology of breast cancer. In Prognostic Factors in Breast Cancer (Edited by Forrest and Kunkler). Longman, London (1968) p. 32. Drasar B. S. and Irving D.: Environmental factors and cancer of the colon and breast. Br. J. Cancer 27 (1973) 167-172. Hill M. J., Drasar B. S., Williams R. E. 0.. Meade T. W., Cox A. G., Simpson J. E. P. and Morson B. C.: Faecal bile acids and Clostridia in patients with cancer of the large bowel. Lancet 1 (1975) 535-539. Reddy B. S. and Wynder E. L.: Metabolic epidemioiogy of colon cancer: Faecal bile acids and neutral sterols in colon cancer patients and patients with adenomatous polyps. Cancer 39 (1977) 2533-2539. Moskowitz M., White C., Barnett R. N., Stevens S., Russel E., Vargo D. and Floch M. H.: Diet, Faecal bile acids and neutral sterols in carcinoma of the colon. Dig. Dis. Sci. 24 (1979) 746-751. Mudd D. G., McKelvey S. T. D., Norwood W., Elmore D. T. and Roy A. D.: Faecal bile acid concentrations of patients with carcinoma or increased risk of carcinoma in the large bowel. Gut 21 (1980) 587-590.
394
ROBERT W. OWENet al.
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