Fat, fibre and cancer risk in African Americans

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Received 23 May 2014 | Accepted 20 Jan 2015 | Published 28 Apr 2015

DOI: 10.1038/ncomms7342

Fat, bre and cancer risk in African Americans and rural Africans

Stephen J.D. OKeefe1, Jia V. Li2, Leo Lahti3,4, Junhai Ou1, Franck Carbonero5,w, Khaled Mohammed1, Joram M. Posma2, James Kinross2, Elaine Wahl1, Elizabeth Ruder6, Kishore Vipperla1, Vasudevan Naidoo7, Lungile Mtshali7, Sebastian Tims3, Philippe G.B. Puylaert3, James DeLany8, Alyssa Krasinskas9,Ann C. Beneel5, Hatem O. Kaseb1, Keith Newton7, Jeremy K. Nicholson2, Willem M. de Vos3,4,10,H. Rex Gaskins5 & Erwin G. Zoetendal3

Rates of colon cancer are much higher in African Americans (65:100,000) than in rural South Africans (o5:100,000). The higher rates are associated with higher animal protein and fat, and lower bre consumption, higher colonic secondary bile acids, lower colonic short-chain fatty acid quantities and higher mucosal proliferative biomarkers of cancer risk in otherwise healthy middle-aged volunteers. Here we investigate further the role of fat and bre in this association. We performed 2-week food exchanges in subjects from the same populations, where African Americans were fed a high-bre, low-fat African-style diet and rural Africans a high-fat, low-bre western-style diet, under close supervision. In comparison with their usual diets, the food changes resulted in remarkable reciprocal changes in mucosal biomarkers of cancer risk and in aspects of the microbiota and metabolome known to affect cancer risk, best illustrated by increased saccharolytic fermentation and butyrogenesis, and suppressed secondary bile acid synthesis in the African Americans.

1 Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.

2 Department of Surgery and Cancer and Centre for Digestive and Gut Health, Institution of Global Health Innovation, Imperial College, London SW7 2AZ, UK.

3 Laboratory of Microbiology, Wageningen University, Wageningen 6703 HB, The Netherlands. 4 Department of Veterinary Bioscience, University of Helsinki, Helsinki, Finland. 5 University of Illinois at UrbanaChampaign, Champaign, Illinois 61801, USA. 6 Division of Sports Medicine and Nutrition, School of Health and Rehabilitation Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA. 7 University of KwaZulu-Natal, Durban, South Africa. 8 Division of Endocrinology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA. 9 Division of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA. 10 RPU Immunolbiology, Department of Bacteriology and Immunology, University of Helsinki, Helsinki 00014, Finland. w Present address: Department of

Food Science, University of Arkansas, Fayetteville, Arizona 72704, USA. Correspondence and requests for materials should be addressed to S.J.D.O.K. (email: mailto:[email protected]

Web End [email protected] ).

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In the west, colon cancer is the second leading cause of cancer death. It aficts B150,000 Americans, 250,000 Europeans and 1 million people worldwide annually. Nearly one third will

die1. Colonoscopy has permitted early detection and recent studies have shown this to be associated with a reduction in mortality rates, but overall impact has been small, particularly among African Americans who shoulder the greatest burden of the disease in the United States. Colon cancer is one of the westernized diseases, with an incidence in Americans of African descent of 65:100,000 compared with o5:100,000 in rural

Africans. Migrant studies, such as those in Japanese Hawaiians, have demonstrated that it only takes one generation for the immigrant population to assume the colon cancer incidence of the host western population2. Although the change in diet is most probably responsible for this3, migration changes many other aspects of the environment. For example, cigarettes, chemicals, infections and antibiotics might be equally responsible for the change in colon cancer risk. To focus on the importance of diet in African Americans and to explore the hypothesis that colon cancer risk is determined by the inuence of the diet on the microbiota to produce anti- or pro-neoplastic metabolites, we have performed a series of investigations between African Americans and rural South Africans4,5. First, we observed that their diets were fundamentally different in preparation, cooking and composition. Animal protein and fat intake was two to three times higher in Americans, whereas carbohydrate and bre, chiey in the form of resistant starch, were higher in Africans. On colonoscopy, African Americans had more polyps and higher rates of mucosal proliferation, measured by Ki67 epithelial cell staining, conrming its potential use as a biomarker of cancer risk6. These differences were shown to be associated with profound differences in the microbiota (Americans dominated by genus Bacteroides and Africans by the genus Prevotella) and metabolic phenotype. Notable differences included higher levels of starch degraders, carbohydrate fermenters and butyrate producers and their metabolites in Africans, and higher levels of potentially pathogenic proteobacteria (Escherichia and Acinetobacter) and bile acid deconjugators and their products in Americans5. As there is extensive experimental evidence that the products of bre fermentation, in particular butyrate, are anti-inammatory and anti-neoplastic79, and that the products of bacterial bile acid conjugation, secondary bile acids, are carcinogenic10, these ndings suggested two potential mechanisms for diet-associated cancer risk: the protective effect of dietary bre in increasing butyrogenesis and the promotional effect of dietary fat on stimulating bile acid synthesis by the liver.

We know from the results of two recently published human studies that modications of the bre and fat content of the diets

have a major impact on the colonic microbiota within a few days11,12. Here we provide critical information on the functional signicance of these microbiota changes on the colonic mucosa by switching the fat and bre proportions of the diet in our two high and low colon cancer risk study populations. Employing the study design outlined on Table 1, we showed the anticipated increase in saccharolytic fermentation and butyrogenesis, and suppression of secondary bile acid synthesis produced by switching African Americans to a high-bre, low-fat diet for 2 weeks, were associated with a signicant reduction in colonic mucosal inammation and proliferation biomarkers of cancer risk. In stark contrast, the diet switch in rural Africans to a high-fat, low-bre diet resulted in reverse changes in all these parameters.

ResultsChanges in the colonic mucosa following diet switch. Measurements made in the home environment (HE) conrmed our preceding studies that the dietary intakes were quite different, with animal protein and fat intake being two to three times higher in Americans and bre intake, chiey in the form of resistant starch, being higher in Africans4 (Supplementary Fig. 1 and Supplementary Tables 13). On colonoscopy, adenomatous polyps were found and removed in nine Americans and no Africans. Measurements of mucosal epithelial proliferation rates by Ki67 staining of biopsies from ascending, transverse and descending colon, all showed signicantly higher levels in African Americans than in Africans (Fig. 1 and Supplementary Fig. 2), again supporting its use as a biomarker of cancer risk. The dietary switch was well tolerated by both populations and body weights were all maintained with 2 kg. Remarkably, the switch decreased proliferative rates in African Americans to levels lower than those of Africans at baseline, whereas rates increased in Africans to levels greater than African Americans at baseline (Fig. 1). In tandem, histological (Supplementary Tables 5ad) and immunohistochemical markers of inammation (Fig. 1b,c) used, namely CD3 intraepithelial lymphocytes and CD68

lamina propria macrophages, increased on switching Africans to the high-fat diet and decreased in Americans given the high-bre diet, thus parallelling the measured changes in mucosal proliferation.

Changes in colonic microbial metabolism following diet switch. The reciprocal changes in mucosal proliferation and inammation between Africans and Americans were associated with reciprocal changes in specic microbes and their metabolites that support our hypothesis that the mechanism whereby diet

Table 1 | Study design, time schedule and sampling.

HE study DI(at home) (in-house)

Day 0 7 14 15 22 29 Period ED 1 HE 1 HE 2 DI 1 DI 2 ED 2

Fecal samples X X X X X X Colonic evacuates X X Breath H2, CH4 X X X X X X

Bloods X X Mucosal biopsies X X Dietary observations XXX X X X X X X X X X X X X X X

DI, dietary intervention periods; ED 1, initial endoscopy days; ED 2, nal endoscopy days; HE, home environment.

The Xs corresponding to dietary evaluation indicate the frequency of dietary recordings: three days during the home environment when consuming their usual diets, and daily during the dietary intervention.

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BeforeAfter *

Ki67 proliferation rate

KI 67

CD3

CD68

0 African American

African

IELS per 100 epithelial cells

African American

African

CD68 infiltration grade

2.5

1.5

0.5

0 African American

African

Figure 1 | Colonic mucosal immunohistochemistry of proliferative and inammatory biomarkers. Immunohistochemical analysis of colonic mucosal biopsies taken at colonoscopy with their associated quantitative analysis on the right panel. (a) An illustration of the paired changes in Ki67 staining of epithelial crypt cells in one African American (the rst and second panels) and one rural African (the third and 4th panels left to right) before and after dietary switch. (b) Paired changes in CD3 staining, similar to a. (c) Paired changes in CD68 macrophages in the lamina propria. The bar graphs on the far right summarize the group means.e. results in 20 African Americans and 12 rural Africans. The two-tailed MannWhitney U-test was used for comparisons for non-paired samples and the Wilcoxon rank sum test for paired samples, with Bonferroni correction for multivariate comparisons. Triangles indicate signicant (Po0.05) baseline differences and stars indicate signicant changes induced by diet switch.

affects colon cancer risk is mediated by the microbiota. First, there was strong evidence that butyrogenesis was associated with lower mucosal proliferation, both in Africans consuming their usual high-bre diet and in African Americans switched to a high-bre African-style diet (Figs 1 and 2, and Supplementary Table 6). As a wide variety of microbes have the capacity to produce butyrate, we targeted our analysis on the measurement of microbial genes encoding the nal enzyme in butyrogenesis, namely butyryl-coenzyme-A-CoA transferase13. On their usual diet, Africans had signicantly greater abundances of butyrate synthesizing genes, and higher fecal butyrate concentrations (Fig. 2) and colonic evacuate quantities (Supplementary Table 7), whereas switching them to a western, low-bre diet suppressed each of these parameters. Reciprocally, our measurements of butyrogenesis were lower in Americans consuming their usual low-bre diet and increased after dietary switch to a high-bre African-style diet. Impressively, Africanization of the diet increased total quantities of butyrate in colonic evacuates 2.5 times, whereas westernization reduced quantities by half.

Recognizing that butyrogenesis is a nal common pathway of bre fermentation, which can only be accomplished by multiple microbial metabolic interactions, we examined the simultaneous changes in the group of microbes that remove the major metabolic end product, hydrogen. If hydrogen is allowed to accumulate, it impairs the regeneration of oxidized pyridine nucleotides, which in turn inhibit the fermentation process and butyrogenesis14. The efcient removal of hydrogen by the hydrogenotrophic microbes, methanogens, which bind hydrogen

into non-toxic methane, sulfate-reducers, which incorporate hydrogen into hydrogen sulde, and acetogens, which consume hydrogen to produce acetate and water, is therefore a prerequisite for enhanced butyrogenesis. Our observation that all these microbial groups were indeed in greater abundance, as were their metabolic products, both in Africans consuming their usual high-bre diet and when Americans were switched to the high-bre diet (Fig. 2) well illustrates the importance of microbial partnership in the maintenance of colonic health.

Flint and colleagues15 have provided strong evidence that the most active butyrate producers, for example, Roseburia spp. and Eubacterium rectale and Faecalibacterium prausnitzii are contained within Clostridium groups IV and XIVa. As Africans and African Americans have very distinct microbiota composition at baseline (Supplementary Table 8), we performed a global analysis of the microbiota by 16S ribosomal RNA gene phylogenetic microarray (Human Intestinal Tract Chip (HITChip)) to examine whether these groups were more prevalent with high bre consumption16. Remarkably, these groups were no more apparent in Africans or Americans eating a high-bre diet. In fact, the compositional changes that were specically associated to the diet switch were minor (ten genus-like groups for Africans and two for Americans; Fig. 3a). These observations suggest either dissociation between structure and function or a change in microbial interactions within the community17. On the other hand, we observed stronger cooccurrence patterns between the genus-level taxa in Africans consuming their usual high-bre diet (Fig. 3b), which included

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Butyrate-producers (bcoA) Bile acid deconjugators (baiCD)

Bilophila wadsworthia (tpA)

Log copy numpers per g Log copy numpers per g Log copy numpers per g

10.0

a a b

5.6

7.5

9.6

Log copy numpers per g

5.2

a b

9.5

9.3

7.0

9.0

4.8

6.5

8.5

8.7

4.4

4.0

6.0

8.0

8.4

7.5

3.6

5.5

8.1

7.0

7.8

3.2

5.0

6.5

7.5

2.8

4.5

6.0

NA AA

4.2

7.2

2.4

NA AA

4.0

HE1 HE2 DI1 DI2 HE1 HE2 DI1 DI2

HE1 HE2 DI1 DI2 HE1 HE2 DI1 DI2 HE1 HE2 DI1 DI2 HE1 HE2 DI1 DI2

HE1 HE2 DI1 DI2 HE1 HE2 DI1 DI2

ED1 ED2

Methanogenic archaea (mcrA)

8.4

3.0

a b

Log copy numpers per g

7.8

6.9

a a a

b b

Log copy numpers per g

8.2

7.6

5.40

6.6

8.0

7.4

5.28

6.3

7.8

7.2

5.16

6.0

7.0

5.04

5.7

7.6

5.4

7.4

6.8

4.92

6.6

4.80

5.1

7.2

6.4

4.8

7.0

4.68

4.5

NA AA

6.2

4.56

6.8

4.2

6.0

Sulfate-reducing bacteria (dsrA)

a b

Fusobacterium nucleatum

7.5

9.6

Log copy numpers per g

9.0

7.5

7.0

6.5

8.4

7.0

6.5

6.0

7.8

a a

6.0

5.5

7.2

a a

5.5

5.0

6.6

6.0

5.0

4.5

4.0

5.4

4.0

3.5

4.8

3.5

3.0

HE1 HE2 DI1 DI2 HE1 HE2 DI1 DI2

Acetate changes with diet and time Split by: group

Box plotGrouping variable(s): period Split by: group

200

Fecal short chain fatty acids

22.5

LCA mol g1 feces

180

Fecal bile acids

160

17.5

140

mol g1 feces

120

100

African Americans

12.5

African Americans

Rural Africans

7.5

2.5

ED 1 HE 1 HE 2 DI 1 DI 2 ED 2

ED 1 HE 1 HE 2 DI 1 DI 2 ED 2 ED 1 HE 1 HE 2 DI 1 DI 2 ED 2

ED 1 HE 1 HE 2 DI 1 DI 2 ED 2

Box plot

Propionate changes with diet and time Split by: group

Grouping variable(s): period Split by: group

mol g1 feces

African Americans

Rural Africans

CA mol g1 feces

African Americans

Rural Africans

Butyrate changes with diet and time Split by: group

2.5

Grouping variable(s): period Split by: group

120

DCA mol g1 feces

100

mol g1 feces

African Americans

Rural Africans

ED 1 HE 1 HE 2 DI 1 DI 2 ED 2

Figure 2 | Targeted Analysis of microbial functional genes of specic interest and their metabolites. Box plots with whiskers and outliers summarizing the results for the targeted functional quantitative PCR analysis of microbial genes of interest (a) and measurements of their metabolic products in fecal samples (b) in 20 African American (AA) and 20 rural Africans (NA) at sequential time points during the study. Group median and variability outside the upper and lower quartiles are shown. Measurements were taken before (Table 1: ED1, HE1 and HE2), during (DI1 and DI2) and after (ED2) 2 weeks of dietary change. With regard to the functional gene analysis, bcoA codes for the enzyme butyryl-CoA:acetate CoA-transferase, which is responsible for the last step in butyrate synthesis, mcrA for the enzyme responsible for methanogenesis, baiCD for the 7-a-dehydroxylating enzyme responsible for secondary bile acid production, and tpA for taurine:pyruvate aminotransferase. LCA, lithocholic acid; CA, cholic acid; DCA, deoxycholic acid. As data were not normally distributed, the Kruskal

Wallis test was used to evaluate the signicance of the group median differences across the different time points shown on the box plots. In the upper panel, comparison with baseline ED1, a signies no signicant change and b a signicant change. In the lower panel for the short-chain fatty acids acetate, propionate and butyrate, and the secondary bile acids LCA and DCA, and primary bile acid CA, there were no signicant differences between time points ED1, HE1 and HE2, while ED2 was consistently different (Po0.05, for details, see Supplementary Table 6). This, together with Supplementary Fig. 6, illustrates no signicant effect from colonoscopy and a profound effect from the dietary change.

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these potential butyrate producers, for example, E. rectale et rel. and Clostridium symbiosum et rel., and bacteria associated with complex carbohydrate utilization, for example, Oscillospira guillermondii et rel. Reduction in bre consumption led to opposite associations (Fig. 3b, bottom right). Reciprocally, high-bre feeding in Americans was associated with a shift from correlations between Bacteroides and potential butyrate-producing groups (Roseburia intestinalis et rel. and C. symbiosum et rel.) towards stronger co-occurrence patterns including Firmicutes that are typically associated with complex carbohydrate fermentation (Fig. 3b, top right).

Global changes in the metabonome following diet switch. We have described metabonomics and the associated term, metabolomics, as the multiparametric metabolic responses of complex systems to perturbations through time18. Specically, metabonomics addresses such phenotypic changes at the level of small-molecule metabolites and maps these processes using appropriate analytical and statistical processes. Recognizing the extreme complexity of the microbiota and its ability to produce a wide array of other metabolites that could affect mucosal health, we performed compositional and pathway analysis19 on fecal water and urine samples by 1H NMR (Figs 4 and 5). Perhaps one of the most striking ndings of this study was the dramatically higher quantities and diversity of metabolites in fecal water in Africans consuming their usual diet compared with African Americans (Fig. 4a). Although the higher levels of products of saccharolytic fermentation were expected from our targeted analysis, the higher quantities of proteolytic fermentation products were not. The only signicantly more abundant fecal metabolite identied in the Americans at baseline, and in Africans after westernization of their diets, was choline. This is highly signicant, as recent studies have shown that choline is extensively metabolized by the microbiota to trimethylamine20 that is absorbed and metabolized by the liver to trimethylamine-N-oxide, which is strongly atherogenic21, providing yet another link between westernized diets, the microbiota and westernized diseases. Evidence that this metabolic pathway was indeed stimulated in Africans given the western diet was the observed increase in urine trimethylamine-N-oxide (Table 2). Reciprocal changes in urinary metabolites derived from microbial metabolism of green vegetables, for example,, N-acetyl-S-

methyl-L-cysteine sulfoxide, were also observed following diet switch, suggesting that some of the reduction in mucosal proliferation in Americans could also have been related to the effects of increased phytochemical consumption in the Africanized diets22. The urinary spectra obtained from African Americans following the change to a high-bre diet also revealed a marked increase in an unrecoginized metabolite (Fig. 4a, left upper panel), possibly a nicotinamide breakdown product, whose character and function have yet to be determined.

The dietary intervention caused, in tandem with the microbiota changes, substantial modications in the metabolic phenotypes of both groups (Table 2, Fig. 4a), with Africans demonstrating less metabolic variation and Americans becoming more diverse. This is probably best appreciated from the metabolic pathway analysis (Fig. 5b,c) of stool and urine samples from both groups, with colour coding identifying the signicant baseline differences, based on integration of the enzymes of bacterial groups identied by HITChip microarray16, host metabolic enzymes, and fecal and urinary metabolites by 1H NMR associated with reactions from the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database. The lower two panels illustrate the signicant shifts in metabolic pathways identied in fecal samples after dietary switch, showing mirror image changes reecting the above, most

notably involving reductions in short-chain fatty acids and increases in glycosylated proteins and choline metabolism in Africans, and the unexpected increases in amino acid metabolism in Americans. Consistent with the results of our targeted analyses, the effects on saccharolytic fermentation were reciprocal, with increases in acetate and lactate in Americans and reductions in Africans. Unlike the results obtained from targeted gas chromatography shown of Fig. 2, the increases in butyrate and propionate measured by global 1H NMR spectroscopy did not achieve statistical signicance, probably possibly due to sample preparation differences between GC and NMR. Again, contrary to expectation, fecal concentrations of colonic branched-chain amino acids, which are likely to be derived from proteolytic fermentation, decreased in Africans given the high-protein western diet for unclear reasons.

Global microbemetabolite associations. To investigate this, we performed a partial correlation analysis between genus-level microbes and urine and fecal metabolites (Fig. 4b), adjusted for nationality and diet. Although we cannot biologically explain all associations (for example, propionate correlates with enter-obacteriaceae and Streptococcus, which are not known to produce propionate), one striking feature was that the metabolite butyrate was exclusively associated with microbial groups that are known to contain butyrate producers, well exemplied by R. intestinalis et rel, E. rectale et rel and C. symbiosum et rel, which are shown in the co-occurrence networks in Fig. 3b, to be enhanced when high-bre diets were consumed by both population groups.

High-fat, low-bre diets and toxic microbial metabolites. It is unlikely that butyrogenesis can alone explain diet-associated cancer risk. Our preceding studies had shown that the higher-fat diet of African Americans was associated with a greater abundance of fecal microbial genes that encode for one of the key enzymes responsible for deconjugation of bile acids (the baiCD gene encodes for the 7a-dehydroxylating enzyme23) as well as their metabolic products, secondary bile acids5. Secondary bile acids have been shown to be carcinogenic in experimental models and multiple human studies have shown that their fecal levels are associated with cancer risk10,24,25. Furthermore, there is substantial experimental evidence that the carcinogenic potential of these metabolites is potentiated by colonic butyrate deciency2629. Once again, we show that there were reciprocal changes in both baiCD gene abundance and the secondary bile acids, lithocholic and deoxcholic acid, after diet switch, with levels increasing in Africans given the high-fat, western-style diet and reductions in Americans fed the low-fat, traditional African diet (Fig. 2, lower panel). Although the use of these functional genes may not cover all the enzymatic pathways of interest, they do provide legitimate tracers for them30. Remarkably, Africanization reduced colonic evacuate secondary bile acids by 70% and westernization increased them by 400% (Supplementary Table 7).

Dietary fat may inuence colon cancer risk through other pathways as illustrated by a recent study in an Il10 / mouse model, which showed that a high-fat diet cultivated an increase in the abundance of Biophila wadsworthia, a member of the Desulfovibrionaceae family, which generates hydrogen sulde via taurine respiration, leading to acute inammation31. Exogenous hydrogen sulde is a potent genotoxin in vitro32. Finally, although no strong evidence has yet been found to indict a specic microbe in colonic carcinogenesis, a recent study found that Fusobacterium nucleatum was enriched in human colon cancer tissue, and suggested that the microbe might promote colorectal neoplasia progression through recruitment

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Bacteroidetes

Clostridium cluster IV

FDR

Bacteroides fragilis et rel. 0.10 Allistipes et rel. 0.12 Bacteroides uniformis et rel. 0.12

Clostridium orbiscindens et rel. <0.01

Clostridium cluster XI

Clostridium cluster XIVa

Clostridium cluster XVI

Uncultured clostridiales

Anaerovorax odorimutans et rel. 0.12

Clostridium colinum et rel. 0.09 Clostridium nexile et rel. 0.10 Dorea formicigenerans et rel. 0.16

Butyrivibrio crossotus et rel. 0.08 Ruminococcus obeum et rel. 0.14

= Home diet = Intervention diet

Native Africans

Eubacterium biforme et rel. 0.10

Uncultured II

Clostridiales 0.09

0.0 1.5

1.0Relative abundance (%)

Clostridium cluster XIVa

0.5

2.0

2.5

3.0

= Home diet = Intervention diet

African americans

0.0 1.5

1.0Relative abundance (%)

FDR

0.5

2.0

2.5

3.0

Butyrivibrio crossotus et rel.

Enterobacter aerogenes et rel.

Desulfovibrio et rel.

Uncultured selenomonadaceae

Burkholderia

Akkermansia

Ruminococcus callidus et rel.

Ruminococcus obeum et rel.

Staphylococcus

Oxalobacter formigenes et rel.

Ruminococcus obeum et rel. Staphylococcus

Oxalobacter formigenes et rel. Proteus et rel.

Clostridium colinum et rel.

Lactobacillus gasseri et rel.

Clostridium colinum et rel.

Clostridium cellulosi et rel.

Ruminococcus lactaris et rel.

Lactobacillus gasseri et rel.

Bacteroides stercoris et rel.

Clostridium cellulosi et rel.

Clostridium orbiscindens et rel.

Anaerostipes caccae et rel.

Sporobacter termitidis et rel.

Allistipes et rel.

Prevotella tannerae et rel.

Ruminococcus gnavus et rel.

Clostridium symbiosum et rel.

Bacteroides plebeius et rel.

Allistipes et rel.

Prevotella tannerae et rel.

Leminorella

Anaerostipes caccae et rel.

Akkermansia

Bacteroides stercoris et rel.

Aquabacterium

Bacteroides uniformis et rel.

Uncultured selenomonadaceae

Ruminococcus lactaris et rel.

Sporobacter termitidis et rel.

Tannerella et rel.

Bacteroides intestinalis et rel.

Clostridium orbiscindens et rel.

Enterobacter aerogenes et rel.

Ruminococcus gnavus et rel.

Clostridium symbiosum et rel.

Roseburia intestinalis et rel.

Butyrivibrio crossotus et rel.

Westerndiet

Uncultured bacteroidetes

Tannerella et rel.

Leminorella

Proteus et rel.

African Americans

Bacteroides uniformis et rel.

African diet

Burkholderia

Aquabacterium

Desulfovibrio et rel.

Mitsuokella multiacida et rel.

Bacteroides plebeius et rel.

Mitsuokella multiacida et rel.

Weissella et rel.

Lachnospira pectinoschiza et rel.

Uncultured bacteroidetesRoseburia intestinalis et rel.

Catenibacterium mitsuokai et rel.

Anaerotruncus colihominis et rel.

Lactobacillus salivarius et rel.

Oscillospira guillermondii et rel.

Ruminococcus callidus et rel.

Prevotella tannerae et rel.

Lactobacillus catenaformis et rel.

Campylobacter

Clostridium difficile et rel.

Coprobacillus catenaformis et rel.

Burkholderia

Weissella et rel.

Lachnospira pectinoschiza et rel.

Helicobacter

Oscillospira guillermondii et rel.

Eggerthella lenta et rel.

Proteus et rel.

Helicobacter

Eggerthella lenta et rel.

Proteus_et_rel.Clostridium stercorarium et rel.

Clostridium symbiosum et rel.

Eubacterium rectale et rel.

Collinsella

Clostridia

Eubacterium rectale etrel.

Native africans

Dorea formicigenerans et rel.

Catenibacterium mitsuokai et rel.

Anaerotruncus colihominis et rel.

Coprobacillus catenaformis et rel.

Clostridium symbiosum et rel.

Campylobacter

Lachnobacillus bovis et rel.

Clostridium difficile et rel.

Africandiet

Ruminococcus callidus et rel.

Prevotella tannerae et rel.

Western diet

Lactobacillus catenaformis et rel.

Clostridium stercorarium et rel.

Lactobacillus salivarius et rel.

Lachnobacillus bovis et rel.

Dorea formicigenerans et rel.

Figure 3 | The impact of diet switch on microbiota composition and co-occurrence networks. (a) An overview of signicantly altered genus-like bacterial groups (FDRo0.2; linear model) following the 14-day diet change. Average fraction of total HITChip signal (proxy for relative abundance) over the samples for each comparison. Black and grey squares indicate the average abundances at home- and intervention diet, respectively. (b) The microbiota genus-like group co-occurrence networks of Africans and African Americans under both dietary regimens for genus-like groups. Groups with correlation differences 41 before and after the diet and at least |r|40.5 are shown. Positive correlations are indicated with green lines; negative correlations with red lines. Identically coloured nodes indicate network modules with three or more co-occurring groups. Grey nodes represent modules of less than three co-occurring groups or those not represented by a module. The sample sizes are 20 African Americans and 17 rural (Native) Africans.

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Urine

Feces

0.8

0.7

O-PLS coefficients (a.u.)

0.6

African American Native African

0.6

0.5

0.4

0.3

0.2

0.1

9 1H p.p.m. 1H p.p.m.

1H p.p.m. 1H p.p.m.

8.5

7.5

6.5

3.5

2.5

1.5

8.5

7.5

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0.75

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Figure 4 | The impact of diet switch on fecal and urinary metabolites and their correlation with fecal microbiota. (a) 1H NMR fecal and urinary spectra obtained from African Americans (AA) and native Africans (NA) during the home environment period (HE) and post dietary intervention (DI). Metabolites distinguishing the two groups are labelled either above the line (higher after dietary intervention) or below (higher in home environment), and the colour represents the correlation coefcient (r2) of the metabolite with the groups. (b) Two-way clustered heatmap of partial correlation between phylotypes assigned to genus-level taxa whose representation in the fecal microbiota (x axis) of African American (AA) and rural Africans (NA), and relative metabolite concentrations in urine and feces (y axis). P-values from correlation were adjusted for multiple testing using the BenjaminiHochberg FDR. Given a large number of tests performed, a cutoff value of 0.3 was used to dene the signicance denoted by . This level follows the Food and Drug

Administration guidelines for bioanalytical biomarkers and was chosen because of the relatively low number of samples it allows for less false negatives. Fecal metabolites (upper compartment) demonstrated a higher number of signicant correlations with fecal microbiota compared with urinary metabolites (lower compartment). E. rectale et rel., Lachnospira pectinoschiza et rel., R. intestinalis et rel. and Lactobacillus bovis et rel. correlate strongly with butyrate, acetate and formate. Adjustments were made for country (binary vector with 0 AA and 1 NA) and diet/stage (0 HE 2 and 1 DI 2, see Table 2) in

calculating the partial correlations).

of proinammatory tumour-inltrating immune cells33. Alternatively, members of our group have hypothesized that fusobacteria may promote genotoxity by their ability to convert cysteine to hydrogen sulde34. Figure 2 (upper panel) shows that the presence and activity of both of these microbes may have played a role in the changes in mucosal biomarkers: the high-bre, low-fat dietary intervention in African Americans was associated with a signicant decrease of B. wadsworthia, whereas the low-bre, high-fat intervention in Africans was associated with an increase of F. nucleatum.

DiscussionWe have shown in individuals from high-risk and from low-risk cancer populations that changes in the food content of bre and fat had remarkable effects on their colonic microbiota and metabonome within 2 weeks, and, critically, that these changes were associated with signicant changes in mucosal inammation and proliferation. Although we cannot claim from our results that these changes in mucosa will result in changes in the development of cancer, there is good experimental evidence that increased epithelial proliferation predicts neoplastic change, because it

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(All)

(AA) (NA)

Tricarboxylic acid (TCA) cycleCoenzyme A metabolism Purine and pyrimidine metabolism

Bile acid metabolism Sulphur metabolism

Branch-chain amino acid metabolism Urea cycle and related metabolites

Pathways

Metabolites Higher after African diet

TCA anaplerotic metabolism Aromatic and indole compoundsGlycolysis and amino sugar metabolism

Higher in African home environment

Higher after African-American diet

Lipid and fatty acid related metabolism

Higher in African-American home environment

1-carbon and related metabolism

Nicotinamide metabolism Lysine metabolism

Higher in urine after African-American diet, higher in faeces after African diet

Figure 5 | The impact of diet switch on fecal and urinary metabolic reaction networks. (ac) Metabolic reaction networks of metabolites found differentially expressed between different dietary comparisons, created using the MetaboNetworks software19 using information from the Kyoto Encyclopaedia of Genes and Genomes (KEGG) on the reactions and enzymes within the biological system. The network shows links between metabolites if the reaction entry in KEGG indicates a main reactant pair and the reaction is either mediated by (1) an enzyme linked to human genes, (2) an enzyme linked to genes from identied bacterial groups using the HITChip or (3) it is part of a spontaneous process. The colour of the metabolites indicate whether the metabolite is found in higher concentrations in the urine/feces of individuals consuming an African diet (orange), higher in individuals consuming an African American diet (blue), higher in the African American home environment (light red), higher in the African home environment (green) or whether the metabolites is not signicantly associated with any comparison but is part of the metabolic network (white). Two metabolites (alanine and formate) are found with opposite associations in feces and urine (Table 2), these are therefore represented with a grey background colour. A table with full names for each of the abbreviated metabolite names can be found in Supplementary Table 11. The background shading indicates different interconnected pathways. In the top part of the legend, the colour for each pathway, as represented in the gure, is shown. The bottom part of the legend, with black boxes around text box, indicates the association of each metabolite. (a) Global network of changes (All) observed in fecal water and urine, and in any comparison of the different groups. (b) Network of fecal metabolites found differentially expressed in African Americans before and after the dietary switch (AA). (c) Network of fecal metabolites found differentially expressed in Africans before and after the dietary switch (NA).

increases the risk of development of DNA mutations due to the higher rate of exposure of sensitive proliferating cells to luminal carcinogens. The evidence for the use of proliferation biomarkers in humans is based on the known increase in the percentage of epithelial cells engaged in DNA synthesis (measured as proliferation index) in all premalignant conditions of the gastrointestinal tract, as well as the observed expansion of the

proliferative zone, causing a shift of the proliferating cells from the base to the surface of the crypt3537. There is also substantial evidence that chronic inammation increases cancer risk38 and it has been observed in human studies that colon cancer is increased vefold in patients with chronic ulcerative colitis38 and reduced 50% by anti-inammatory drugs39. In keeping with this, it was noteworthy that our two biomarkers on mucosal inammation,

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Table 2 | Fecal and urinary metabolites.

(a) Fecal metabolites

African HE (N 19)

versus DI (N 20)

Q2Y 0.59; R2X 29.8% Q2Y 0.74; R2X 26.8% Q2Y 0.66; R2X 37.2% Q2Y 0.37; R2X 30.7%

r P q r P q r P q r P q Valine 0.99 0.77* o0.001 0.002 0.82w o0.001 0.003

Leucine 0.97 0.75* o0.001 0.003 0.75w 0.001 0.02 Isoleucine 1.01 0.78* o0.001 0.001 0.77w o0.001 0.01

Glutamate 2.36 0.82w o0.001 o0.001 0.84w o0.001 o0.001 Alanine 1.48 0.7* 0.003 0.007 0.79w o0.001 0.009

Glycine 3.57 0.72* 0.002 0.005 0.82w o0.001 0.002 Tyrosine 6.91 0.74* o0.001 0.003 0.81w o0.001 0.001

Butyrate 0.89 0.67* 0.007 0.01 0.71* o0.001 0.006 Propionate 1.06 0.75* o0.001 0.002 0.71* o0.001 0.006

Valerate 1.29 0.73* 0.001 0.004 0.66* 0.005 0.01 Acetate 1.93 0.74* o0.001 0.003 0.7w 0.03 0.006 0.75* o0.001 0.005

Lactate 1.34 0.75* o0.001 0.002 0.77w o0.001 0.009Formate 8.45 0.69* 0.002 0.009

Choline 3.20 0.78y o0.001 0.001 0.668 0.006 0.01 Uracil 5.81 0.78* o0.001 0.001

Hypoxanthine 8.21 0.74* o0.001 0.003 0.69* 0.002 0.008 6-Aminosalicylicacid8

7.01 0.85w o0.001 o0.001 0.77w o0.001 0.005

Fumarate 6.53 0.69* 0.002 0.008 Phenylacetate 3.54 0.81* o0.001 o0.001b-Xylose 4.58 0.72* o0.001 0.006 b-Arabinose 4.53 0.74* o0.001 0.005

Nicotinate 8.62 0.71* o0.001 0.005 Threonine 4.26 0.77* o0.001 0.002

(b) Urinary metabolites

Chemical shift (1H p.p.m.)

HE African American (N 15) versus African

(N 19)

DI African American (N 14) versus African

(N 20)

African American HE (N 15) versus DI

(N 14)

Chemical shift (1H p.p.m.)

African HE (N 12) versus

DI (N 12)

Q2Y 0.41; R2X 26.6% Q2Y 0.86; R2X 36.4% Q2Y 0.51; R2X 20.8% Q2Y 0.83; R2X 27.8%

r P q r P q r P q r P q

HE African American (N 8) versus African

(N 12)

DI African American (N 7) versus African

(N 12)

African American HE (N 8) versus DI

(N 7)

N-acetyl S-methyl-L-cysteine sulfoxidez

2.78 0.8* 0.003 0.16 0.93w o0.001 o0.001 0.93w o0.001 0.03 0.8* o0.001 0.007

3-Methylthiopyruvic acid sulfoxidez

2.81 0.81* 0.002 0.14 0.94w o0.001 o0.001 0.95w o0.001 0.009 0.77* o0.001 0.008

3-Methylthiolactic acid sulfoxidez

2.76 0.88w o0.001 o0.001 0.83w 0.002 0.12 0.79* 0.001 0.01

S-methyl-L-cysteine sulfoxide

2.84 0.84w 0.003 0.13

p-Cresyl sulfate 2.35 0.7* 0.03 0.28Phenylacetylglutamine 2.27 0.71* 0.02 0.263-Hydroxyisovalerate 1.27 0.78y 0.005 0.18 0.858 o0.001 0.004 2-Methylpropan-2-olz 1.14 0.91w o0.001 o0.001 0.89w o0.001 0.07

Alanine 1.49 0.88 0.003 0.02 0.75y 0.03 0.33Valine 0.99 0.858 o0.001 0.003

Isoleucine 0.93 0.888 o0.001 o0.001 Carnitine 3.23 0.888 o0.001 0.001 0.918 o0.001 o0.001

O-acetylcarnitinez 2.15 0.918 o0.001 o0.001 0.918 o0.001 o0.001

N6-acetyllysinez 1.99 0.9w o0.001 0.01

Citrate 2.53 0.85w o0.001 0.003 0.71w 0.04 0.39 0.71* 0.006 0.04 Creatine 3.94 0.738 0.004 0.03

Creatinine 4.07 0.82w 0.001 0.01 0.77* o0.001 0.007 Trigonelline 4.44 0.9w o0.001 o0.001 0.86w 0.002 0.124-Hydroxyhippurate 6.97 0.81w 0.003 0.023-Hydroxymandelate 6.92 0.87w o0.001 0.002

Dimethylamine 2.73 0.88* o0.001 0.07 0.79w 0.01 0.25 0.78* 0.001 0.01 Trimethylamine-N-oxide

3.27 0.768 0.02 0.07

Choline 3.23 0.848 o0.001 0.004 Formate 8.46 0.798 0.002 0.01

N-methyl-2-pyridone-5-carboxamidez

6.66 0.938 o0.001 o0.001

DI, dietary intervention; HE, home environment.

Parts (a) and (b) show signicantly changed fecal and urinary metabolites in each pairwise comparison (for example, African American versus African during HE, African American at HE versusDI). Representative chemical shift of each metabolite is provided. The r-values are Pearsons correlation coefcient from the correlation of the metabolite concentrations with classication (for example, African Americans versus Native Africans). P- and q-values represent the signicance of the metabolite changes and false discovery rate-adjusted P-values76, respectively.*Higher in HE: Africans.

wHigher in dietary switch: African Americans.

zTentative assignment.yHigher levels of metabolites in HE: African Americans.

8Higher in dietary switch: Africans.

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CD3 intraepithelial lymphocytes and CD68 lamina propria

macrophages responded in the same direction as our Ki67 proliferative biomarker. CD3 intraepithelial lymphocytes provide a measure of T-cell activation and, therefore, the pivotal balance between immune activation and quiescence in response to luminal antigens, such as bacteria40,41, whereas CD68 lamina propria macrophages play an active role in the persistence of chronic inammation42 and neoplastic progression43,44. Interestingly, the microbial metabolite, butyrate, was shown recently to reduce the responsiveness of lamina propria macrophages to commensal bacteria45, suggesting that the dietary change in Americans may have resulted in a decrease in both macrophage numbers and their activity.

Our study revealed two unanticipated colonic mucosal ndings in rural Africans consuming their usual baseline diets. First, despite the signicantly lower epithelial proliferation and population colon cancer rates, mucosal inammation detected endoscopically, histologically and by immunohistochemistry was more common and pronounced compared with high-risk African Americans (Fig. 1). Second, despite the lower protein intake and lower plasma amino acid levels (Supplementary Table 9), proteolytic fermentation was also increased, releasing metabolites that are known to be inammatory and pro-carcinogenic46. The explanation for these ndings is unclear, but may be linked to the high prevalence of chronic parasitic infestations in rural Africans causing inammation and exudation of mucinous proteins. Of note, schistosoma were incidentally identied in two subjects and a tapeworm segment in another (Supplementary Fig. 3). Intestinal protozoa have been hypothesized to stimulate immunosurveillance against colon cancer47. Clearly, further focused investigations of these paradoxical ndings are needed, but possible explanations involve the complex associations between inammation, immunosurveillance and carcinogenesis, and the ability of butyrogenesis to arrest the progression of inammation to neoplasia. In murine models, lymphocytes can suppress carcinogenesis as part of the process of immunosurveillance, and in patients with tumours, specic intra-tumour lymphocyte markers for Th1 polarization, and cytotoxic and memory T cells have been shown to suppress growth and metastasis, a process termed immunoediting48,49. Furthermore, T-cell inltration may suppress or enhance tumorigenesis, depending on the type, density and location of the immune cells. There is substantial experimental evidence, recently reviewed by us7,8, that the major products of saccharolytic fermentation, acetate, propionate and butyrate have potent anti-neoplastic properties50, but butyrate is unique in its maintenance of colonic mucosal health and defense. First, it is the preferred energy source for colonocytes51. Second, it has a remarkably wide variety of antineoplastic properties, best exemplied by its function as a histone deacetylase inhibitor52, its capacity to downregulate the key canonical Wnt-signalling pathway linked to colonic carcinogenesis53 and its ability to reduce the burden of carcinogens, such as bile acids28,29,54 and red meat products26. Further studies are needed to explore the intriguing possibility that increased saccharolytic fermentation and butyrogenesis may counter the recognized proliferative properties of T-helper type 17 T cells55.

Although we have focused on the potential for a high-bre and high-fat diet to modify metabolic pathways that have been shown experimentally to have an impact on mucosal biomarkers of cancer risk, it must be stressed that a change in one aspect of an otherwise isocaloric diet will inevitably result in a change in another. Consequently, the associated change in animal protein or digestible carbohydrate might also be responsible for the observed mucosal changes. Be that as it may, our results provide fairly substantial evidence for a powerful inuence of butyrate on

mucosal health and raise hope that an increase in bre consumption, together with a moderation in fat intake, may reduce the disproportionately high incidence of colon cancer in African Americans and indeed in most westernized communities. Clearly, these suggestions now need to be conrmed by longitudinal studies. With regard to bre, total quantities may be critical, as bre supplementation at E35 g per day has generally failed to reduce polyp recurrence in clinical trials56 and recent experimental studies demonstrate that a threshold effect of butyrate availability determines its inhibitory effect on proliferation52. Furthermore, human studies by Burkitt57 concluded that total bre intake needs to exceed 50 g per day (as in our study) to prevent colon cancer. Our results also raise serious concern that the progressive westernization of African communities may reduce butyrogenesis, allowing their high basal state of chronic inammation to progress through proliferation to neoplasia38, culminating in the emergence of colon cancer as a major health issue58.

Methods

Study design. First, the study was submitted to the University of Pittsburgh and University of KwaZulu-Natal Institutional Review Boards for approval. We employed a unique design (Table 1 and Supplementary Note 1), where 20 healthy middle-aged African Americans and 20 rural Africans (Supplementary Tables 10: Demographics) from the same communities previously studied5 were studied rst for 2 weeks in their own HE, eating their usual food (HE study), and then again in-house while they were fed the intervention diet for 2 weeks (dietery intervention) study). Consequently, each subject served as his/her own control, which is important given the known wide individual variation in colonic microbiota composition. We chose intervention diets that were at the same time palatable and contained reverse quantities of bre and fat, such that African Americans would be given African style foods increasing their average bre intake from 14 to 55 g per day and reducing their fat from 35% to 16% of total calories, whereas Africans were given a western-style diet reducing their bre from 66 to 12 g per day and increasing their fat from 16% to 52% (Supplementary Tables 13). Cogniscient of the problems of compliance to acute dietary change and the accuracy of dietary recall to estimate actual intakes within the community, we elected to perform all the dietary intervention studies in-house, where meals could be prepared and given under close supervision. With African Americans, participants were housed the University of Pittsburgh Clinical Translational Research Center, and with rural Africans we employed in a rural lodging facility, close to their homes, with full kitchen facilities. Body weights were maintained within 2 kg by adjusting food quantities while keeping the overall macronutrient composition the same. The sampling schedule is given on Table 1, showing that fresh fecal samples were taken at three intervals during the HE study and again three times after the diet switch. Colonoscopy was performed to identify latent disease, polyps, or cancer, to obtain 3 h colonic evacuates for analysis and biopsies for biomarkers of cancer risk4 at the beginning of the HE study and end of the dietery intervention study. Full details of the menus, cooking methods and total dietary compositions are given under Supplementary Tables 2, 3 and 4ac, and Supplementary Note 2).

Subjects and recruitment. Age- and sex-matched healthy volunteers, with the age range 5065 years, were randomly selected from the African American population in the Pittsburgh region of Pennsylvania and from the rural native South Africans from the rural Kwazulu region. We collaborated with Dr Stephen Thomas, Director of Minority Studies at the University of Pittsburgh School of Public Health, to recruit healthy African American volunteers from the Pittsburgh region, and also advertised the study with the approval of our Institutional Review Board in public areas. In South Africa, volunteers were be recruited through advertisements placed in public community centres, for example, post ofces, town halls, civic centres and through the iZulu Community Health Center. Appropriate compensation (as advised by Minority studies and the iZulu Community and ratied by the University of Pittsburgh and KwaZulu-Natal) for time and testing was paid to volunteers for participation.

Screening. Informed signed consent was taken from each participant. All African volunteers could understand English, but a nurse-translator participated in the consent process to ensure proper understanding of the details of the research procedures. Screening was performed in Pittsburgh at the Clinical Translational Research Center and in Africa at Ngwelezana Hospital outpatient clinic, Empangeni, KwaZulu-Natal, South Africa. A detailed medical history was rst taken. With rural Africans, a local bilingual nurse acted as an interpreter. A 20-ml blood sample was taken for full blood count, ESR, electrolytes and urea, albumin, alkaline phosphatase, AST and bilirubin. If the results were normal and if they satised the eligibility criteria, they were invited to participate in the study.

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Subject eligibility. Details are given under Supplementary Note 1. Inclusion criteria were as follows: healthy volunteers, from GI standpoint between 40 and 65 years (age at which colon cancer screening/colonoscopy is recommended in this population) and body mass index between 18 and 35 kg m 2 (Supplementary

Discussion). Exclusion criteria were as follows: participants pre-colonoscopy were ineligible if they had a history of familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, inammatory bowel disease or invasive cancer within 5 years before enrollment (h/o adenomatous polyps acceptable). In addition, ineligible participants were individuals with known renal, hepatic or bleeding disorders; previous GI surgery resulting in disturbed gut function due to of loss of bowel or altered anatomy, or any form of chronic GI disease resulting in disturbed gut function, diarrhoea and malabsorption; and individuals with antibiotic use within the past 12 weeks (Supplementary Discussion), current steroids use or with diabetes. Exclusion criteria post colonoscopy were detection of previously unrecognized ulceration (with depth and 40.5 cm), stricture, severe inammation and polyps 41 cm diameter or cancer.

Fecal, colonic and mucosal sampling and colonoscopy. To synchronize the measurements of the microbiota, the metabolome and the colonic mucosa, the sampling was tied to the preparation for and conduct of a colonoscopy, as previously described4, at baseline while on their usual diet (ED1, Table 1), and then again at the conclusion of the dietary change (ED2, Table 1). In this, fresh fecal samples are collected before colonic evacuation and immediately frozen at 80 C

and the total colonic contents are collected for 3 h during evacuation with a simple polyethylene glycol solution. In this, 2 l of polyethylene glycol (60 g l 1, average molecular weight 3350 g/mol) solution was consumed rapidly as possible over 30 min. We avoided using the commercial preparation Golytely, as it contains sodium sulfate, which is known to disturb the microbiota composition. Our experience had shown that the quality of bowel preparation with this technique was similar to the more conventional overnight bowel washout. The mucosa health status was assessed by visualization and biopsy. Polyps, when encountered, were removed per standard practice and biopsies were taken from normal mucosa from the proximal (caecum/ascending colon), mid (transverse) and distal (sigmoid) colon at 25 cm from the anal verge. Mucosal samples for immunohistochemistry were collected in formalized saline and for gene expression in RNAlater (Qiagen, Germantown, MD) before being stored at 80 C.

Measurement of mucosal biomarkers. Histology. Colonic mucosal biopsies were obtained by colonoscopy before and after dietary switch from three different sites (ascending, transverse and descending) and stored in 10% buffered formalin. Later, the biopsy samples were embedded in parafn and 5-mm sections were cut and stained with either routine haematoxylin and eosin (H&E) or immunohisto-chemical stains (see below). The histologic ndings on the H&E-stained sections were evaluated by one blinded, experienced gastroenterological histopathologist (AK). Measurements focused on the numbers of inammatory cells and eosinophils in the lamina propria and the numbers of intraepithelial lymphocytes. Scoring of the H&E-stained section was done as shown in Supplementary Table 5a. Any pathologic nding, such as the presence of parasitic organisms, was recorded.

Immunohistochemistry. Slides for CD3 and Ki67 staining were deparafnized at 60 C for 2 h. To inhibit endogenous peroxidase, the slides were pre-treated using 3% hydrogen peroxide/methanol at room temperature (RT) for 10 min, followed by antigen retrieval with 0.2% pepsin solution (P7012, Sigma, 3050 Spruce St, St Louis, MO, USA) at 37 C for 10 min. Serum Free Protein Block (X0909, Dako, 6392 Via Real, Carpinteria, CA 93013, USA) was used at RT for 10 min. The slides were drained and incubated with primary antibodies CD3 and Ki67 (A0452, 1:100, rabbit polyclonal, Dako; MIB-1, 1:100, mouse monoclonal, Dako), respectively, at RT for 1 h. Secondary detection was applied using Immpress universal antibody Polymer detection kit (MP-7500, Vector Labs, 30 Ingold Road, Burlingame, CA 94010, USA) at RT for 30 min. The slides were stained with DAB substrate kit (SK-4100, Vector Labs) for 10 min and counter stained using Shandon Hematoxylin (6765015, Thermo Scientic, 81 Wyman St, Waltham, MA 02451, USA).

Slide staining for CD68 was done on a Ventana Benchmark Ultra slide stainer. Deparafnized slides were pretreated using the ultra CC1 (950-224, Ventana, 1910 Innovation Park Dr, Tucson, AZ 85755, USA) for 24 min for antigen retrieval. Slides were incubated with the primary antibody CD68 (M087601, 1:100, mouse monoclonal, Clone PG-M1, Dako,) for 32 min RT. The Optivew DAB kit (760-700, Ventana) was used for secondary detection.

Quantication of immunohistochemical staining. Counting of the proportions of positive staining cells using light microscopy at 400 magnication was

performed by a single investigator (KM), under blinded conditions. To assess inter-observer variability, 40 slides were randomly selected and recounted by a second senior pathologist (AK), showing a concurrence of 88% for Ki67 and

80% for CD68 densities.

Ki67. The proportion of Ki67-positive staining cells were counted in well-oriented crypts (average/slide 8, range 414). Ki67 proliferation rate was dened as the

number of Ki67 cells divided by the total number of crypt cells and were

expressed as percentage. The differences were found to be the same in the total crypt and in the upper crypt; hence, only the total crypt proportions are reported here.

CD3. Only CD3 staining intraepithelial lymphocytes were counted in a representative area of at least 300 epithelial cells. The density of intraepithelial lymphocytes was expressed as an index of number of CD3-positive lymphocytes per 100 epithelial cells.

CD68. The number of CD68-positive cells (macrophages) within the lamina propria were counted and graded on a scale from 1 to 3: grade 1 (none/rare), grade 2 (scattered supercial collections) and grade 3 (strong, diffuse or band-like inltrate in the supercial lamina propria) as shown in Fig. 1c.

Targeted analysis of fecal and colonic microbes and metabolites of special interest. Details of the materials and methods used for the collection, preparation and analysis of fecal samples for targeted analysis of microbes of special interest (real-time quantitative PCR) and their metabolites (Agilent Technologies 6890N Network GC System with a ame-ionization detector for short-chain fatty acids and Shimadzu HPLCmass spectrometry for quantication using electrospray ionization in negative ion mode by monitoring the (MH) ion for bile acids) in

African Americans and rural Africans have been previously published5. Justication for the use of the BcoA functional gene for butyrate production is that Flints group in Aberdeen have demonstrated that although there are a number of different metabolic pathways that use different enzymes that culminate in butyrate synthesis, butyryl-CoA:acetate CoA-transferase, the product of the BcoA gene, is responsible for the last step in butyrate synthesis in the vast majority of intestinal butyrate producers13. Similarly, there are other microbial enzymes that participate in bile acid deconjugation, but Wells et al.23 demonstrated that there was a good correlation between human fecal bacterial dehydroxylating activity measured by fecal dilution assay and their PCR assay for the baiCD gene, which encodes the key enzyme responsible for the bile acid 7a-dehydroxylation pathway.

Plasma amino acids. Fasting blood concentrations were measured in extracted plasma by reverse-phase C-18 precolumn derivatization HPLC (AccQ Tag Ultra

derivatization, Waters, Milford, MA) as previously described5.

Statistical analysis. Statistical analysis of the group differences in continuous variables was conducted using SPSS 16.0 (SPSS Inc.). The signicance of group differences for normally distributed data was assessed with unpaired and paired Students t-tests. The non-parametric data were analysed with a MannWhitney U-test or KruskalWallis one-way analysis of variance (ANOVA) by ranks for unpaired data and Wilcoxon signed-rank tests for paired data. The signicance of the association was evaluated with Spearmans rank correlation test. A level of Po0.05 was accepted as statistically signicant. Data are presented as means s.e.

Complex microbiota and metabonome data were analysed by several multivariate ordinations detailed below (principal component analyses and non-metric multidimensional scaling), KruskalWallis independent tests and multivariate ANOVA with Bonferroni correction.

Global analysis of the microbiota composition and diversity. We chosethe HITChip phylogenetic microarray for the global proling of microbiota composition. It has been demonstrated that the HITChip analysis of fecal samples provides highly concordant results concerning the microbiota composition when compared with 16S rRNA gene or metagenome sequencing5962, as it allows deep proling of phylotypes at high resolution, down to o0.1% relative abundance, corresponding to a duplicated set of 100,000 pyrosequencing reads per sample with very high reproducibility (498%)59 and at considerably lower cost.

HITChip analysis. DNA was isolated from fecal or colonic samples and subsequently used for phylogenetic proling of the intestinal microbiota using the HITChip phylogenetic microarray16. Standardized quality control was maintained through our library of a duplicated set of 3,631 probes targeting the 16S rRNA gene sequences of over 1,000 intestinal bacterial phylotypes. Briey, the full-length 16S rRNA genes were amplied, and PCR products were transcribed in vitro into RNA, labelled with Cy3 and Cy5, and fragmented. Hybridizations were performed in duplicate and data were extracted from microarray-scanned images using Agilent Feature Extraction software, version 10.7.3.1 (http://www.agilent.com

Web End =http://www.agilent.com). Array normalization was performed as previously described, using a set of custom R scripts (http://r-project.org

Web End =http://r-project.org) and stored in a custom MySQL database (http://www.mysql.com

Web End =http:// http://www.mysql.com

Web End =www.mysql.com ). Duplicate hybridizations with a Pearson correlation 498%

were considered for further analysis. Microbiota proles were generated by preprocessing the probe-level measurements with minimummaximum normalization and the frozen Robust Probabilistic Averaging probe summarization62,63 into three phylogenetic levels: level 1, dened as order-like 16S rRNA gene sequence groups; level 2, dened as genus-like 16S rRNA gene sequence groups (sequence similarity 490%); and level 3, phylotype-like 16S rRNA gene sequence groups (sequence

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similarity 498%). In the present work, we primarily focus on the genus-level (level2) variation. Signicance of the differences between the time points (Fig. 3a) were estimated based on a (paired) linear model for microarrays (limma) with the threshold of false discovery rate (FDR)o0.2 estimated based on Benjamini

Hochberg procedure and a minimum fold change of 25% (0.1 at the Log10 scale)64.

Microbial co-occurrence network analysis. We constructed the co-occurrence networks between the 130 genus-like bacterial groups based on their logarithmic abundances (HITChip log10 signal) within each treatment group (African Americans and Native Africans; before and after the dietary intervention). For a robust correlation analysis, we applied the SparCC algorithm using 20 iterations and with 50 bootstrap data sets for signicance testing, followed by q-value65 correction of the pseudo P-values from the bootstrap analysis. We focused on the signicant (|r|40.5; qo0.01) correlations between the genus-like groups where a qualitative change (change of sign) in the correlation was observed following the dietary intervention. To ensure robust analysis, we included only the correlations that changed drastically in the intervention, with a difference of 41 between the correlation values before and after intervention (so that the correlations would change for instance from 0.5 to 0.5, or higher). This provided a list of genus-like

bacteria that had signicant changes in their mutual correlation networks following the dietary intervention in either rural African or African American group. To simplify interpretation, we clustered the associated genus-like groups into coherent network modules with complete-linkage hierarchical clustering based on the SparCC correlations. We dened a module as a connected sub-network where the correlations between all genus-like groups within the module are r40.5. Networks were visualized using the network visualization and exploration platform Gephi66. The resulting modules are highlighted in Fig. 3b.

Global analysis of the metabolome: sample preparation for NMR spectroscopic analysis. All urinary samples were thawed at RT and vortexed for 10 s.

A total of 400 ml of urinary sample was thoroughly mixed with 250 ml of 0.2 M sodium phosphate buffer containing 20% D2O, pH 7.4, 0.01% 3-(trimethylsilyl)-[2,2,3,3-2H4]propionic acid sodium salt and 3 mM sodium azide (NaN3). The mixture was subsequently centrifuged at 10,000g for 10 min and 600 ml of super-natant was transferred into an NMR tube with an outer diameter of 5 mm. Approximately 200 mg of wet fecal sample was mixed with 600 ml of H2O (HPLC grade) in a 1.5-ml Eppendorf tube. A cycle of 30-s vortexing, 5-min sonicatingat 4 C and 30-s vortexing was then carried out on the mixture, followed by centrifuging at 10,000g for 10 min. A total of 400 ml of supernatant was added into a1.5-ml Eppendorf tube containing 250 ml of aforementioned sodium phosphate buffer, vortexed and spun at 10,000g for 10 min. An amount of 600 ml of supernatant was put into an NMR tube.

1H NMR spectroscopy. 1H NMR spectra of urinary and fecal water extract samples were acquired using a Bruker 600 MHz spectrometer (Bruker, Rheinstetten, Germany) at the operating 1H frequency of 600.13 MHz at a temperature of 300 K. An NMR pulse sequence (recycle delay -90-t1-90-tm-90-acquisition) and standard parameters were used to obtain standard one-dimensional 1H NMR spectral data as described in Beckonert et al.67

Multivariate statistical data analysis. 1H NMR spectra of urine and fecal extracts were automatically phased, referenced to 3-(trimethylsilyl)-[2,2,3,3-

2H4]propionic acid sodium salt at 1H d0.00 and baseline-corrected using an in-house developed MATLAB script (Dr Tim Ebbels, Imperial College London). The processed NMR spectra (1H d 0 to 10) were imported to MATLAB (R2012a,

MathWorks) and digitized into 20k data points with a resolution of 0.0005 p.p.m. using an in-house developed script. The water peak region in urine (4.75.14 p.p.m.) and fecal water spectra (4.75.18 p.p.m.) were removed to minimize the effect of the disordered baseline caused by water suppression. In addition, regions (1H 00.3) in urine spectra and regions (1H 00.25) in fecal water spectra containing only noise were therefore removed, together with the urea signal in urine (1H 5.486.24). Because of the heavy peak shifting in urinary spectra, a recursive segment-wise peak alignment method was applied, to improve metabolic bio-marker recovery68. Probabilistic quotient normalization was subsequently performed on the resulting data sets, to account for dilution of complex biological mixtures69. Principal component analysis and orthogonal partial least-squares discriminant analysis was carried out with a unit variance scaling method in SIMCA (P 13.0) and MATLAB software. P-values of metabolites were calculated

using ANOVA and FDR was also calculated for each metabolite marker70. For the two-way clustered heatmap (Fig. 4b) between fecal microbiota and metabolite concentrations in urine and feces, the partial correlation was calculated for each phylotype and metabolite. The correlation was adjusted for country and dietary intervention to account for baseline differences. A pFDR cutoff value of 0.3 was used to dene the signicance (denoted by ), to account for the different

numbers of samples in each data set. For each data set (microbiota, fecal metabolites and urinary metabolites), the data were clustered using hierarchical cluster analyses with complete linkage. The optimal number of clusters was chosen as the splitting that maximized the modularity using the correlation as weight of each edge in the network.

Metabolic reaction network generation. Using the freely available Metabo-Networks software19 reactions that occur spontaneously or by means of enzymes linked to human and/or bacterial genomes were identied in the KEGG. All complete genome sequences listed in KEGG that could be associated to the bacterial groups represented on the HITChip were included in creating the database, resulting in a total of 817 bacterial genomes included in the database. Next, the software was used to construct a shortest-path metabolic reaction network between all (fecal and urinary) metabolites signicantly different in any of the different dietary comparisons. From this global network, two sub-graphs were generated for each of the two dietary switches using signicantly associated fecal metabolites. To highlight the differential expression of the metabolites associated with specic pathways, the background shading was added to the graphs to indicate the different interconnecting pathways (Fig. 5ac). The abbreviations and full names of these metabolites can be found in Supplementary Table 11.

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Acknowledgements

We thank Chadd and Kate Bain, and the iZulu Lodge and Projects team in KwaZulu-Natal, for help with the recruitment and housing of the African volunteers, and Dr Iain Thirsk and his surgical endoscopy staff at Ngwelezana Hospital, Empangeni, KwaZulu-Natal, for providing the endoscopic facilities for the study, and Faye Brouard for help with the rural dietary assessments. We acknowledge the help of Dr Robert Branch and the staff of the University of Pittsburgh Clinical Translation and Research Center for help with the dietary switch studies on African Americans. We thank Ms Kayellen Umeakunne for help with the design of the intervention diets. Primary funding for the study was provided by a grant from the National Institutes of Health R01 CA135379 (OKeefe) and CTRC support from UL1 RR024153 and UL1TR000005. The research was also supported by the National Institute for Health Research Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London. We acknowledge the Academy of Medical Sciences, which funded part of the metabolic proling work. Additional funding for the microbiota studies was provided by the Spinoza Award of the Netherlands Organization (de Vos) for Scientic Research, the ERC Advanced Grant 250172 (Microbes Inside) of the European Research Council and the Academy of Finland (grant 141140 to W.M.dV. and grant 256950 to L.L.). The University of Pittsburgh Genomics and Proteomics Core Laboratory (GPCL) produced the mucosal gene expression microarray data.

Author contributions

S.J.D.OK. designed and conducted the human studies, supervised the analysis and evaluation the results, prepared, edited and revised the manuscript. E.G.Z. assisted with study design, managed the global microbiota analysis and interpretation, and assisted with preparation of the manuscript. H.R.G. assisted with study design and execution of the human studies, managed the targeted microbiota analysis and interpretation, and assisted with preparation of the manuscript. J.K. was responsible for writing the text relating to the 1H NMR analysis and for interpreting the spectral data. J.N. provided strategic oversight for the metabonomic analytical strategy and was responsible for data interpretation. J.L. performed the 1H NMR analysis and the multivariate analysis. J.O.

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7342

was responsible for targeted analysis of metabolites, data management, sample distribution and manuscript preparation. L.L. and S.T. conducted the microbiota data analysis and interpretation, and assisted with preparation of the manuscript. P.P. sample coordination and microbiota phylogenetic proling. W.M.D.V. managed, interpreted and supported microbiota analysis. K.M. and H.K. performed the mucosal immunohistochemistry. E.W. (USA), E.R. (USA) and F.B. (S Africa) performed the dietary analysis, meal design and production. K.A.N. and V.N. helped organize the South African studies and endoscopies. K.V. was responsible for the screening, recruitment and management of African Americans dietary switch studies. F.C. performed the targeted microbiota analysis. L.M. supervised the recruitment of African subjects and helped with the dietary exchange studies. A.K. supervised the histological assessments of biopsy samples. A.C.B. coordinated the targeted microbiota analyses. J.D. supervised the targeted metabolite analysis and helped with manuscript preparation. J.P. carried out the metabolic reaction network analysis and interpretation.

Additional information

Accession codes: Microbiome and metabolome data have been deposited in the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad.1mn1n.

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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How to cite this article: OKeefe, S. J. D. et al. Fat, ber and cancer risk in African Americans and rural Africans. Nat. Commun. 6:6342 doi: 10.1038/ncomms7342 (2015).

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Rates of colon cancer are much higher in African Americans (65:100,000) than in rural South Africans (<5:100,000). The higher rates are associated with higher animal protein and fat, and lower fibre consumption, higher colonic secondary bile acids, lower colonic short-chain fatty acid quantities and higher mucosal proliferative biomarkers of cancer risk in otherwise healthy middle-aged volunteers. Here we investigate further the role of fat and fibre in this association. We performed 2-week food exchanges in subjects from the same populations, where African Americans were fed a high-fibre, low-fat African-style diet and rural Africans a high-fat, low-fibre western-style diet, under close supervision. In comparison with their usual diets, the food changes resulted in remarkable reciprocal changes in mucosal biomarkers of cancer risk and in aspects of the microbiota and metabolome known to affect cancer risk, best illustrated by increased saccharolytic fermentation and butyrogenesis, and suppressed secondary bile acid synthesis in the African Americans.

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Title

Fat, fibre and cancer risk in African Americans and rural Africans

Author

O'keefe, Stephen J D; Li, Jia V; Lahti, Leo; Ou, Junhai; Carbonero, Franck; Mohammed, Khaled; Posma, Joram M; Kinross, James; Wahl, Elaine; Ruder, Elizabeth; Vipperla, Kishore; Naidoo, Vasudevan; Mtshali, Lungile; Tims, Sebastian; Puylaert, Philippe G B; Delany, James; Krasinskas, Alyssa; Benefiel, Ann C; Kaseb, Hatem O; Newton, Keith; Nicholson, Jeremy K; De Vos, Willem M; Gaskins, H Rex; Zoetendal, Erwin G

Pages

6342

Publication year

2015

Publication date

Apr 2015

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Nature Publishing Group

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20411723

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Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms7342

ProQuest document ID

1676199646

Fat, fibre and cancer risk in African Americans and rural Africans

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