TOXICOLOGICAL SCIENCES, 164(2), 2018, 453–464

doi: 10.1093/toxsci/kfy102
Advance Access Publication Date: April 20, 2018
Research Article

Aflatoxin B1 Disrupts Gut-Microbial Metabolisms of

Short-Chain Fatty Acids, Long-Chain Fatty Acids, and

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Bile Acids in Male F344 Rats
Jun Zhou,*,† Lili Tang,*,† Jincheng Wang,*,† and Jia-Sheng Wang*,†,1
†
*
Interdisciplinary Toxicology Program; and Department of Environmental Health Science, College of Public
Health, University of Georgia, Athens, Georgia 30602
1
To whom correspondence should be addressed. Fax: (706) 542-7472. E-mail: [email protected].

ABSTRACT
In this study, male F344 rats were orally exposed to aflatoxin B1 (AFB1) at 0, 5, 25, and 75 lg/kg for 4 weeks. Rat feces were
collected from 2 to 4 weeks following exposure and were assessed for gut-microbiota-dependent metabolites. Gut-
microbiota-related organic acids were quantitated in the feces using 2-nitrophenylhydrazine derivatization coupled HPLC-
profiling method which was validated and showed good reliability, accuracy and sensitivity. After 2-week exposure, AFB1
significantly reduced the levels of fecal short-chain fatty acids (SCFAs) with an over 70% reduction in the high-dose group
(75 lg/kg). Mixed-effects model revealed an inverse correlation between AFB1 dose and fecal levels of SCFAs, but no
significant time effect was found. When compared with the control, oral exposure to middle-dose AFB1 (25 lg/kg) resulted
in remarkable elevations of fecal cholic acid (2.18-fold), linoleic acid (cis-9, cis-12–18:2) (11.3-fold), pentadecanoic acid (15: 0)
(3.68-fold), pyruvic acid (4.56-fold), and 3-phenyllactic acid (3.74-fold), but deoxycholic acid level was reduced by 41% in the
low-dose group (5 lg/kg). These results demonstrated the disruptions of several important gut-microbiota metabolic
pathways, including the synthesis of SCFAs, pyruvic acid related pathways, metabolisms of amino acids, bile acids and
long-chain fatty acids, which may further affect host digestive efficiency, energy supply, intestinal immunity, production of
neurotransmitters, and enterohepatic cross-talk. Our study suggests that the impairment of gut-microbiota-dependent
metabolism may contribute to pathological mechanisms of AFB1-induced adverse health effects.

Key words: Aflatoxin B1; gut-microbiota; long-chain fatty acids; microbial metabolism; short-chain fatty acids.

Aflatoxins (AFs) are a class of food-borne mycotoxins mainly assessment of AFB1 contamination in human food and animal
produced by Aspergillus flavus and Aspergillus parasiticus (Kumar feed has been a global concern for food safety and public health
et al., 2016). These toxigenic fungi commonly contaminate soil, (Henry et al., 1999; Torres et al., 2015). On the other hand, re-
and colonize on the surface of cereals, especially for maize, and markable efforts have been made to develop novel prevention/
groundnuts, once humidity (>17.5%) and temperature (>24 C) intervention strategies against AFB1-induced adverse health
meet their growth needs (Trenk and Hartman, 1970). Such envi- effects, including liver cancer risks and growth/developmental
ronmental conditions have made the tropical area more suscep- disorders in high-risk and vulnerable populations (Mitchell
tible for the food contamination and human exposure to AFs, et al., 2014; Xue et al., 2016).
especially in the low- and middle-income developing nations Human gastrointestinal tract harbors a complex microbiota
(Qian et al., 2013a). Aflatoxin B1 (AFB1) is widely recognized as that contains more than 100 trillion microbes with over 400 spe-
the most harmful AF, due to its potent toxicity, genotoxicity, cies and carries 150 times more genes than the human genome
and carcinogenicity as well as acute aflatoxicosis in animals (Qin et al., 2010). The gut-microbiota constantly provides host
and human populations (Kew, 2013; Qian et al., 2013b; Wang with hundreds of micronutrients and functional metabolites,
and Groopman, 1999). Accordingly, the detection and which actively participate into the host enterohepatic

C The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology.
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 454 | AFB1 DISRUPTS GUT-MICROBIAL METABOLISM

cross-talk, as well as the physiological regulations of many and 75 lg AFB1/kg body weight (b.w.) per day, respectively.
organs and systems (Ursell et al., 2014). In recent years, next- DMSO was used as vehicle solvent. The details of animal proto-
generation sequencing technologies have uncovered all kinds of col were reported in earlier publications, together with body in-
intricate connections among gut-microbiota, dietary composi- dexes, histopathological assessment and AFB1-Lys
tion and host health (Chakraborty et al., 2010; Holmes et al., pharmacokinetic data (Mohammadagheri et al., 2016; Qian et al.,
2012). In this 3-way relationship, oral exposure to xenobiotics or 2013b, 2014, 2016). Briefly, animals were daily administered
dietary composition could lead to the alteration of gut- with AFB1 by gavage for 4 weeks. From the second week to the
microbiota, and the changes of gut-microbiota may further in- fourth week, rat feces were daily collected, and weekly pooled
fluence host health in a significant way (Brown and Hazen, for each group. All fecal samples were stored in 80 C freezer.
2015). Emerging evidences have demonstrated the causative Animal husbandry and care, AFB1 dosing protocol, and sample
links between gut-microbial microbiome/metabolome and a se- collection were approved and in strict accordance with the
ries of health problems in host, eg, obesity, metabolic syn- requirements and regulations of the Institutional Animal Care
drome, nonalcoholic fatty liver disease (NAFLD), colon cancer, and Use Committee at the University of Georgia.
inflammatory bowel disease (IBD), and cardiovascular disease

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(Flint et al., 2012; Holmes et al., 2012; Lee and Hase, 2014; Louis Sample quenching and extraction. Sample extraction procedure
et al., 2014; Ursell et al., 2014). Therapeutic manipulation of gut- was similar to what previously published with modifications
microbiota has also exhibited the potential to mitigate a num-  ndez Bort et al., 2014). Cold methanol
(de Jonge et al., 2012; Herna
ber of metabolic diseases such as obesity, type-2 diabetes (80 C)-based quenching and extraction were applied to the fe-
mellitus, IBD and NAFLD, most probably by modifying gut- cal samples for sample pretreatment. The purpose of using cold
microbiota-dependent metabolites, which are either derived methanol was to avoid the loss of volatile compounds, and also
from food by gut-microbiota, or the endogenous metabolites of because methanol is a solvent chemically appropriate for the re-
gut-microbes (Kootte et al., 2012; Schulberg and De Cruz, 2016). action of 2-NPH derivatization (Peters et al., 2004; Torii et al.,
We have previously performed 16S rRNA analysis and found 2010; Winder et al., 2008). To perform sample extraction 200 mg
the compositional change of fecal microbiome in F344 rats fol- rat feces was transferred to the Mobio PowerLyzer tube with
lowing repeated oral exposure to AFB1 (Wang et al., 2016). preloaded glass beads (0.1 mm i.d.). One milliliter of cold metha-
Through 16S rRNA sequencing technique, notable enrichment nol was immediately added into the tube, and fecal pellet was
of Clostridiales spp. and depletion of Lactobacillales spp. were gently crushed using a glass pestle. After grinding, 0.5 ml cold
found in the rat feces. In the work presented here, the potential methanol was slowly added to wash the pestle. Then the tube
impact of such compositional changes on host health at meta- was capped tightly and fastened on a rotary vortex to undergo
bolic level was further explored by examining a group of fecal 20 min vortex at maximum level using a Vortex-Genie 2 Mixer
organic acids that are highly associated with gut-microbiota. (Scientific Industries). During vortex, sample tube was put back
The studied metabolites include acetic acid, lactic acid, propi- on ice for 2 min in every 5 min, and finally underwent centrifuga-
onic acid, butyric acid, valeric acid, hexanoic acid, cholic acid, tion at 12 000 rpm for 10 min to spin down cellular debris. A
deoxycholic acid, pentadecanoic acid (15: 0), 3-phenyllactic acid, volume of 100 ml supernatant was transferred to an Eppendorf
pyruvic acid, and linoleic acid (cis-9, cis-12-18:2). The metabo- tube, and 50 ml internal standard (2-ethylbutyric acid) stock solu-
lism of these organic acids heavily depends on the metabolic tion was spiked into the supernatant to achieve a concentration
pathways and community structure of gut-microbiota, and also of 1 mg/ml, which was used to compensate technical variabilities.
play important roles in host physiology and global metabolic
pathways. 2-NPH derivatization. To perform derivatization, 150 ml sample
extract (with internal standard added) was mixed with 45 ml
derivatization solution which was freshly prepared by mixing
MATERIALS AND METHODS 15 ml EDC solution (0.05 g/mL H2O), 15 ml 2-NPH solution (12.5
Chemicals and reagents. Pyridine, 2-nitrophenylhydrazine (2- mg/ml methanol) and 15 ml 3% pyridine in methanol (v/v). After
NPH), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydro- mild vortex, the tubes were transferred to water bath at 60 C for
chloride (EDC), 2-ethylbutyric acid, acetic acid, propionic acid, 60 min. The tubes then were allowed to stay in room tempera-
butyric acid, valeric acid, hexanoic acid, cholic acid, pentadeca- ture for 5 min and went through brief centrifugation in order to
noic acid, 3-phenyllactic acid, pyruvic acid, linoleic acid, deoxy- collect the liquid left on the tube wall. All sample vials were
cholic acid, bisphenol A, hippuric acid, heptadecanoic acid, kept in 4 C sample cooling tray and the analysis was finished
AFB1, and dimethyl sulfoxide (DMSO) were all purchased from within 24 h.
Sigma-Aldrich Inc. (St Louis, Missouri). AFB1 stock solution
(25 mg/ml) was prepared in DMSO and diluted to appropriate High-performance liquid chromatography analysis. An Agilent 1200
treatment concentrations upon using. All other reagents and High-performance liquid chromatography (HPLC) system, con-
analytical solvents, methanol, acetonitrile, and water were pur- sisted of a degasser, a quarterly pump, an autosampler, a diode-
chased at the highest grade commercially available from array detector, and a fluorescence detector, was used to perform
Honeywell (Morris Plains, New Jersey). HPLC-profiling analysis. The chromatographic separation was
conducted in a Nucleosil C18 reversed-phase column (250  4
Animal treatment. Male Fischer 344 rats (100–120 g) were pur- mm i.d.; ES industries, New Jersey) with particle size of 5 lm
chased from Harlan Laboratory (Indianapolis, IN, USA). The ani- and pore diameter of 120 Å. The injection volume was 100 ml
mal housing environment was under controlled light/dark cycle and flow rate was kept at 1 ml/min. Column oven temperature
(12:12 h) with a temperature of 22 C 6 2 C and relative humidity was set as 40 C. Mobile phase A was pH 4.5 acidified water ad-
of 50%–70%. Purified AIN 76A diet and tap water were main- justed by hydrochloric acid. Mobile phase B was acetonitrile.
tained every day. Upon arrival, animals were allowed for one The gradient eluting condition was: 90% A to 80% A in 0–12 min;
week of environmental acclimation. One hundred male F344 80% A to 70% A in 12–20 min; 70% A to 60% A in 20–30 min; 60%
rats were divided into 4 groups and were gavaged with 0, 5, 25, A to 45% A in 30–41 min; 45% A to 10% A in 41–43 min; then
 ZHOU ET AL. | 455

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Figure 1. HPLC-profiling chromatograms of fecal extracts from control (upper) and exposure group (lower) after 2-NPH derivatization. 2-ethyl butyric acid was used as
internal standard (IS). The detection channel of DAD is 400 nm with a reference channel as 510 6 60 nm. Down-regulated organic acids are labeled on the upper panel,
whereas up-regulated organic acids are labeled on the lower panel. Specific retention time and relevant information are available in Table 1.

keeping at 10% A in 43–58 min; finally, from 10% A to 90% A in analyte amount measured in the extract of nonspiked feces) 
58–61 min for re-balance. The detection channel is 400 nm by 100/(amount of spiked analyte) (Han et al., 2013b).
DAD, with reference wavelength at 510 6 60 nm. The represen-
tative chromatogram is shown in Figure 1. Lower limit of detec- 16S rRNA analysis. Briefly, total fecal genomic DNA which con-
tion (LLOD), regression standard curves, as well as the other tains 16S rRNA was extracted using QIAamp DNA stool mini kits
necessary quantitative parameters used for HPLC-profiling (QIAGEN, Valencia, California). A 2-step Quadruple-index PCR
analysis are listed in Table 1. Short-chain fatty acids (SCFAs) method was used to prepare the 16S rRNA gene libraries accord-
were recovered using the recovery rates averaged from the feces ing to Klindworth et al. (2013). Sequencing of these 16S rRNA frag-
spiked with SCFA standards of approximately 50%, 100%, and ment libraries was performed in the Georgia Genomic Facility
200% of their levels in control group (Supplementray Table 1). (University of Georgia, Athens, Georgia) using the Illumina MiSeq
The concentrations of other interested analytes were deter- with v2 500 cycle chemistry, resulting in paired-end 250 base
mined using the recovery rates of structurally close standards reads to obtain approximately 30 000 reads per sample.
which have similar or close structure to the analytes. The 16S rRNA fragment amplified in this study is from site
358 to 784 under Escherichia coli system of nomenclature
Specifically, the recovery rate of hippuric acid was used to re-
(Klindworth et al., 2013). The raw paired-end, demultiplexed se-
cover phenyl acids (PAs); heptadecanoic acid was used to re-
quence read was merged using FLASH 1.2.9 in Geneious 8.1 soft-
cover long-chain fatty acids (LCFAs), and bisphenol A was used
ware (Biomatters Inc, San Francisco, California). All internal
to recover bile acids. Further, 2-ethylbutyric acid was used to
tags, base spacers, and locus-specific primers of merged
eliminate the technical variabilities, since it has similar struc-
sequences were trimmed and sequences outranged 400–450
ture with SCFAs. Bisphenol A was used as standard to calculate
base-pairs were discarded. Outputs from Geneious 8.1 were
recoveries for bile acid and derivatives because it is considered
quality filtered using QIIME pipeline (Quantitative Insights Into
to have close structure with estradiol, which was used as inter-
Microbial Ecology) (Caporaso et al., 2010). Representative
nal standard for quantitative analysis of bile acid (Junichi et al.,
sequences for each operational taxonomic units (OTUs) were
1978; Rubin, 2011). And no other commercially available com-
compared with the Greengene 16S rRNA gene database 13-8 re-
pound can be chromatographically separated with bile constitu-
lease (DeSantis et al., 2006) using uclust algorithm with the simi-
ents for the calibration of recovery using current method. larity threshold of 90%. The top 3 database hits that matched
the above representative sequences for each OTU were selected.
Method validation and optimization. Methanol blanks were spiked
with SCFA standards to generate test solutions with concentra- Statistics and software. Data normality examination, homogene-
tions of approximately 50%, 100%, and 200% of the actual SCFA ity test, 1-way ANOVA, and Welch’s t test, were all performed
amounts measured in the sample extracts. The test solutions using SPSS 22. Levene statistic was used to test homogeneity of
were derivatized using 2-NPH and EDC and were immediately variances and Welch-Brown-Forsythe statistic was used to test
used for HPLC-profiling analysis. The analytical precision of the the equality of means. Tukey’s test was used for post hoc analy-
method was validated based on: (1) interday coefficient of varia- sis in ANOVA. When data failed to follow normality of distribu-
tion (CV) of the peak intensities of SCFAs at 3 spike levels in 3 tion, Kruskal-Wallis H test was applied to replace 1-way
consecutive days, with one bunch performed per day; (2) inter- ANOVA. Mixed-effects model regression was performed using
assay CV of the peak intensities of SCFAs at 3 spike levels in 7 STATA 14.1. Pearson’s correlation analysis, and construction of
consecutive assays; (3) intra-assay CV of the peak intensities of heat map and hierarchical tree were performed using R. Mann-
SCFAs at 3 spike levels, with 4 repeats conducted at each level. Whitney U test was used to compare the differences of fecal or-
Analytical accuracy was examined using recoveries with CV, ganic acids (except for SCFAs) when dose effect was the only
and the formula to calculate recovery rate is: recovery % ¼ (ana- factor being analyzed, with p value < .05 considered to be statis-
lyte amount measured in the extract of standard-spiked feces – tically significant.
 456 | AFB1 DISRUPTS GUT-MICROBIAL METABOLISM

Table 1. Analytical Parameters of HPLC-Profiling Analysis Used for the Measurement of Interested Fecal Metabolites

Component Category RT* Detection Channel Regression (X, AUC; Y, ng/ll) R2 Linear Range ng/ll LLOD ng/ll

Acetic acid SCFA 14.9 400 nm y ¼ 0.0063x  0.3726 0.9993 0.016–64.8 0.008
Propionic acid SCFA 19.6 400 nm y ¼ 0.021x  0.9273 0.999 0.07–143 0.03
Butyric acid SCFA 25.1 400 nm y ¼ 0.0256x  0.4994 0.9991 0.078–79.5 0.04
Valeric acid SCFA 31.5 400 nm y ¼ 0.0208x  0.4974 0.999 0.054–56.1 0.03
Hexanoic acid SCFA 37.6 400 nm y ¼ 0.0309x  0.356 0.9994 0.074–75.6 0.04
Lactic acid SCFA 13.8 400 nm y ¼ 0.0244x  0.2351 0.9991 0.11–14.33 0.05
Pyruvic acid Alpha-keto acid 41.3 400 nm y ¼ 0.0166x þ 0.7136 0.9997 6.2–500 0.19
2-Ethylbutyric acid IS for SCFA 34.2 400 nm y ¼ 0.1662x - 0.4527 0.9991 0.56–1138 0.28
Niacin PA 22.1 210 nm y ¼ 0.0313x  6.1768 0.9954 1–430 0.25
3-Phenyllactic acid PA 31.2 400 nm y ¼ 0.1003x  0.7134 0.9994 4.7–300 0.58
Hippuric acid IS for PA 26.3 400 nm y ¼ 0.6161x þ 2.8275 0.9996 4.45–570 2.25
Cholic acid SA 45.1 400 nm y ¼ 0.1219x  6.6046 0.9930 3.9–250 0.49

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Deoxycholic acid SA 47.1 400 nm y ¼ 0.0371x  4.8658 0.9930 2.5–330 0.64
Cholesterol Sterol 47.4 400 nm y ¼ 0.0686x  2.4795 0.9900 1.95–125 0.98
Bisphenol A IS for SA 35.0 210 nm y ¼ 0.0148x  6.9647 0.992 0.33–685 0.17
Linoleic acid LCFA 50.9 400 nm y ¼ 0.3705x  31.314 0.9948 3.9–1000 3.9
Pentadecanoic acid LCFA 51.2 400 nm y ¼ 0.0636x  0.3641 0.9990 1.95–500 0.5
Heptadecanoic acid IS for LCFA 54.5 400 nm y ¼ 0.1436x  4.5852 0.9952 2.15–275 1.07

The minimum data point in the linear regression range (R2 > 0.999) was noted as LOQ.
Abbreviations: IS, internal standard for quality control; R2, regression coefficient; LLOD, lower limit of detection; LCFA, long-chain fatty acid; PA, phenyl acid; RT, reten-
tion time (min) in chromatogram; SA, steroid acid; SCFA, short-chain fatty acid. The analyte level which generated a signal-to-noise (S/N) ratio of 3 was noted as the
LLOD for that analyte. Niacin and cholesterol were not detected in most sample extracts.

RESULTS feces of AFB1-exposed groups. In the low-dose group, fecal SCFA

levels seemed to be affected by the time of exposure. The fecal
Validation and Optimization of HPLC-Profiling Method levels of acetic acid, propionic acid, butyric acid, lactic acid, va-
Our initial effort was to optimize conditions for fecal sample ex- leric acid and hexanoic acid were 46.6%, 39.9%, 68.4%, 79.9%,
traction and metabolites enrichment. However, centrifugal 95.3%, and 63% of the control after 2 weeks of exposure, but the
evaporation resulted in significant loss of SCFAs (20%–50%) in percentages went to 70.7%, 77.6%, 35.1%, 34.4%, 75.6%, and
the sample extracts, as found by HPLC analysis (data not 86.7% of the control after 4 weeks of exposure, indicating the
shown). For this reason, sample enrichment was avoided during time-effect of AFB1-exposure on SCFA levels in the low-dose
sample preparation. Nonetheless, interested analytes are still group. The fecal levels of SCFAs in the middle- and high-dose
detected from fecal samples. In terms of precolumn derivatiza- groups were generally not affected by the exposure time, except
tion and HPLC-profiling analysis, the validation work included for propionic acid in middle-dose group at 2-week, and lactic
intraassay precision, interassay precision, interday precision, acid and hexanoic acid in high-dose group at 4-week, which
and accuracy. Shown in supplementary Table 1, most values of showed about 50% changes of fecal levels compared with con-
measured metabolites showed CV < 8%. The sensitivity and trol. As shown in Supplementary Table 2, the fecal levels of 6
LLOD were determined for all analyzed metabolites, as shown SCFAs in the middle-dose group, were 17.7%, 31.1%, 26.1%,
in the Table 1. Internal standards were used to confirm the pre- 20.1%, 90.7%, and 19.9% of the control in 2-week, and were
cision and accuracy, and recovery rate was ranged from 33% to 21.6%, 15.3%, 24.6%, 17.2%, 88.9%, and 27.3% of the control in 4-
74% for the all SCFA standards spiked into fecal samples of the week. Similarly, In the high-dose group the 6 SCFAs were 22%,
control and AFB1-dosed rats, with CV < 5%. Using this validated 22.2%, 21.9%, 12.1%, 44.2%, and 20.5% of the control in 2-week,
method, the peak identity and concentrations of interested and were 25%, 34.4%, 17.2%, 6.8%, 42.2%, and 52% of the control
metabolites were further determined from the chromatogram in 4-week. Remarkable changes were found for fecal propionic
of fecal extracts, as shown in Figure 1. Four categories of metab- acid level in middle-dose group, which was reduced from 31.1%
olites were measured in the study: SCFAs, including acetic acid, of control to 15.3% of control from 2- to 4-week; valeric acid in
butyric acid, hexanoic acid, lactic acid, propionic acid, and vale- high-dose group, which was reduced from 12.1% of control to
ric acid; LCFAs, including linoleic acid (cis-9, cis-12-18:2) and 6.8% of control; and lactic acid in high-dose group, which
pentadecanoic acid (15:0), bile acids, including cholic acid and was elevated from 20.5% of control to 52% of control from 2- to
deoxycholic acid, and other metabolites, including 3-phenyllac- 4-week.
tic acid and pyruvic acid (Table 1). To examine the AFB1 dose-, time-, and time  dose interac-
tion effects on fecal SCFA levels, mixed-effects regression model
AFB1 Exposure Affects SCFA Production of Gut-Microbiota was applied to analyze the linear correlation between the AFB1-
Rats were exposed to AFB1 at doses of 0, 5, 25, and 75 lg/kg b.w., dose/time and SCFA levels. As shown in Table 2, significant
which are noted as control, low-, middle- and high-dose groups dose effect and dose  time interaction effect were found.
in the study. As shown in Figure 2 and Supplementary Table 2, Further, Pearson’s correlation analysis was performed to exam-
significant change of fecal SCFA levels was found in AFB1-ex- ine the possible link between the changes of SCFA levels and
posed groups. The measured levels of SCFAs in the untreated the community structure of gut-microbiota. The correlation
control group were comparable over the time course from 2- to results were shown in the hierarchical tree and heat map in
4-week, but notable reduction of acetic acid, propionic acid, bu- Figure 3. Briefly, strains belonging to Firmicutes Clostridiales order
tyric acid, hexanoic acid, and lactic acid were detected in the rat were highly clustered and showed inverse correlation with the
 ZHOU ET AL. | 457

Figure 2. Fecal SCFA levels of rats treated with 0, 5, 25, and 75 lg AFB1/kg b.w. X-axis indicates duration of treatment. Significance of one-way ANOVA or Kruskal-Wallis
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H Test is indicated by string labels: same string indicating p > .05; string with partly overlapped character(s) indicating p < .05; totally different string indicating p < .01.
Error bar indicates standard deviation (n ¼ 5). Specific data are available in Supplementary Table 2.

fecal levels of SCFAs following AFB1-exposure, while and pentadecanoic acid). Specifically, the level of linoleic acid
Lactobacillales Streptococcus and Clostridiales Roseburia, 2 SCFA- was 95.51 6 24.18 ng/mg in the control group, and increased to
producing strains, were depleted in the feces. All 6 SCFAs are 1274.82 6 363.02 ng/mg in the low-dose group and 1079.18 6
correlated in the same cluster of Pearson’s r distance. 760.29 ng/mg in the middle-dose group; the level of pentadeca-
noic acid in the control group was 20.26 6 21.99 ng/mg, and in-
AFB1 Exposure Affects Metabolism of Other Gut-Microbiota creased to 64.76 6 36.57 ng/mg in the low-dose group and 74.60
Dependent Organic Acids 6 53.35 ng/mg in the middle-dose group; the most significantly
We next examined the impacts of AFB1 treatment on a set of altered organic acid was linoleic acid, with over 10-fold increase
key organic acids after 4 weeks of AFB1 exposure, including cho- found in low- and middle-dose groups (Figure 4).
lic acid, deoxycholic acid, 3-phenyllactic acid, pyruvic acid, pen- Oral AFB1 exposure also significantly elevated fecal levels of
tadecanoic acid (15:0), and linoleic acid (cis-9, cis-12-18:2). Oral cholic acid, pyruvic acid, and 3-phenyllactic acid. The level of
AFB1 exposure significantly elevated fecal LCFAs (linoleic acid cholic acid in the control group was 56.15 6 27.15 ng/mg, and
 458 | AFB1 DISRUPTS GUT-MICROBIAL METABOLISM

Table 2. Mixed-Effects Model Analysis Between AFB1-Treatment (Dose, Time and Interaction) and Fecal Levels of SCFAs

Fixed Effect Random Effect

SCFAs Dose SE p Time SE p Interactiona SE p Estimate SE

Acetic acid 0.1103 0.025 <.001 0.1613 0.133 0.227 10.361 2.149 <.001 31.288 6.021
Propionic acid 0.0617 0.015 <.001 0.054 0.082 0.509 6.718 1.301 <.001 11.798 2.270
Butyric acid 0.1442 0.015 <.001 0.0202 0.086 0.921 13.226 1.663 <.001 18.316 3.525
Valeric acid 0.0028 0.001 <.001 0.0007 0.004 0.863 7.900 0.977 <.001 0.026 0.005
Hexanoic acid 0.0179 0.004 <.001 0.0074 0.022 0.740 2.113 0.365 <.001 0.881 0.170
Lactic acid 0.0207 0.003 <.001 0.0228 0.018 0.210 1.280 0.298 <.001 0.586 0.112

a
Estimate of interaction effect resulted by both dose and treatment time on fecal SCFA levels.

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Clostridiales Roseburia
Clostridiales spp.
Lactobacillales Streptococcus

SCFAs

Lactic Acid
Acetic Acid
Propionic Acid
Butyric Acid
Valeric Acid
Hexanoic Acid
denovo207
denovo953
denovo1623
denovo1221
denovo1585
denovo187
denovo621
denovo88
denovo290
denovo383
denovo1034
denovo1391
denovo2273
denovo1392
denovo1761
denovo504
denovo510
denovo1762

Figure 3. Hierarchical cluster tree and heat map to show cross correlations for SCFAs and top 18 significantly altered gut-microbial strains discovered by previous 16s rRNA
data. Data were transferred to fold change of exposure group versus control. Hierarchical clusters are constructed based on Pearson’s r distance. Red-blue color bar indicates
Pearson’s correlation coefficient between 2 correlated components. SCFAs are negatively correlated with the Clostridial Ruminococcaceae strains that are frequently seen
in the stools from patients with Crohn’s disease and obesity. The suppressed strains belong to Lactobacillales and Clostridial Roseburia. Phylogenetic taxa information
can be accessed in reference (Wang et al., 2016). (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.)

increased to 128.46 6 15.35 ng/mg in the low-dose group and low-dose group from 10.18 6 8.69 ng/mg in the control group,
122.60 6 7.32 ng/mg in the middle-dose group; the level of and completely dropped to undetectable level in the middle-
pyruvic acid in the control group was 38.46 6 26.92 ng/mg, and dose group.
increased to 75.57 6 22.18 ng/mg in the low-dose group
and 175.23 6 74.98 ng/mg in the middle-dose group, and the
level of 3-phenyllactic acid in the control group was 28.82 6
DISCUSSION
9.04 ng/mg, and increased to 83.89 6 18.10 ng/mg in the low-
dose group and 107.84 6 74.9 ng/mg in the middle-dose group, Results of this study clearly demonstrated that up to 2-week
respectively. oral AFB1 exposure disrupted metabolism of gut microbiota-
On the other hand, the level of deoxycholic acid was signifi- dependent organic acids, as evidenced by significant reduction
cantly reduced, to about the half level (5.13 6 5.09 ng/mg) in the in fecal level of SCFAs and deoxycholic acid, and significant
 ZHOU ET AL. | 459

Fold = 4.56
Fold = 2.29 Fold = 2.18
p < 0.001 Fold = 11.3
p < 0.001 p < 0.001
p < 0.01
Fold = 13.35
p < 0.001
Fold = 1.96
p < 0.05

Fold = 3.68 Fold = 3.74

Fold = 3.2 p < 0.01 Fold = 0.59 p < 0.01
p < 0.01 p < 0.05
Fold = 2.91
p < 0.01
Not Detected

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Control 5 μg AFB 1 /kg B.W. 25 μg AFB 1 /kg B.W.
Figure 4. Fecal concentrations of cholic acid, deoxycholic acid, linoleic acid, pentadecanoic acid, pyruvic acid, and 3-phenyllactic acid measured from the experimental
groups treated with 0, 5, and 25 lg AFB1/kg b.w. via HPLC-profiling analysis. Nonparametric Mann-Whitney U test was applied for all comparisons (n ¼ 10). Box with
middle vertical line represents 25%, 50%, and 75% percentile of data. Vertical lines of box plots indicate SD, multiplied with 1.5-fold coefficient in order to stretch out
from box.

increases in LCFAs and other organic acids such as pyruvic acid, Galisteo et al., 2008; Morrison and Preston, 2016; Wong et al.,
3-phenyllactic acid, and cholic acid. All these microbial metabo- 2006; Zhao et al., 2006). Acetic acid, butyric acid and propionic
lites play key roles in the metabolism of gut-microbiota and the acid can be produced by gut-microbiota via fermentation of in-
maintenance of host nutrition and health. soluble fibers (Corre ^a-Oliveira et al., 2016; Morrison and Preston,
The detection of trace amounts of SCFAs in complex media, 2016; Torii et al., 2010). SCFAs were mainly produced from the
eg, bio-fluids and fecal extracts, has been reported by several fermentation process of certain strains such as Lactobacillales
studies using HPLC-profiling combined with precolumn deriva- Streptococcus. The aflatoxin-caused reduction in these microbial
tization with 2-NPH (Han et al., 2013a; Miwa et al., 1985; Peters strains (Wang et al., 2015) could eventually affect the fermenta-
et al., 2004), but the application of this method has not yet tion process and cause reduction of SCFAs. Mixed-effects model
reported in AFB1-exposed rat models. The chemical derivatiza- analysis showed that—3 major SCFAs, ie, acetic acid, butyric
tion is usually performed in mild aqueous or alcohol environ- acid and propionic acid were the most significantly affected by
ment, in which carbonyl compounds (carboxylic acid, aldehyde, AFB1-dose and dose  time interaction, but not time of treat-
and ketone) bonded to 2-NPH and form hydrazides. The reaction ment (Table 2). It was demonstrated in our earlier 16S rRNA
is activated by water-soluble EDC which serves as carbodiimide analysis, that the adaption of gut-microbiota community struc-
crosslinker. Before in-lab analysis, method validation was con- ture was featured by the elevation of relative abundances of
ducted to confirm whether the analytical procedure is suitable Clostridiales spp., but decrease of Lactobacillales Streptococcus and
and reliable for a specific analytical task (VanHook, 2016). The Clostridiales Roseburia (Wang et al., 2016). Given that dose-
accuracy and reliability of analytical method were further care- response was also found for specific gut-microbial strains,
fully validated (Supplementary Table 1). The measured values Pearson’s correlation analysis between fecal SCFA levels and
and interclass ratio of SCFAs in our study are comparable with gut-microbial strains was performed to show their correlation.
several other publications (Cummings et al., 1987; Torii et al., We found that strains from Firmicutes Clostridiales, an order asso-
2010; Zhao et al., 2006). ciated with diarrhea in human and other mammals
In this study we found significant inhibitory effects of AFB1- (Suchodolski et al., 2015), were highly clustered, and exhibited
exposure on synthesis of SCFAs, which has not previously inverse correlation with SCFAs. By contrast, the relative abun-
reported. The decrease in SCFAs was consistent with the deple- dances of Lactobacillales Streptococcus and Clostridiales Roseburia
tion of SCFA-producing strains such as Lactobacillales were positively correlated with fecal SCFAs. Both of these
Streptococcus and Clostridiales Roseburia (Duncan et al., 2002; microbes are SCFA-producing strains (Duncan et al., 2002;
Kleessen et al., 1997). SCFAs are a group of beneficial aliphatic Kleessen et al., 1997). The depletion of SCFAs in feces reflected
acids that are mainly produced by the anaerobic bacterial fer- the suppression of microbial fermentation on resistant starches
mentation of resistant starches and insoluble fibers in the gas- and insoluble fibers. This may further result in a wide range of
trointestinal tract of human and other mammals (Brockman, adverse consequences, because the receptors of SCFAs such as
2005). They are structurally constructed by 1–6 carbon atom(s), GPR43, GPR41, OLFR78, and GPR109A, are extensively distributed
including formic acid (C1), acetic acid (C2), propionic acid (C3), in different organs and systems, and are involved in a myriad of
butyric acid (C4), valeric acid (C5), hexanoic acid (C6), and a vari- regulatory axis and pathways, such as mobility of gut epithe-
ety of branched-chain isomers of these acids. A variety of nutri- lium, liver lipogenesis, global immunity, cell cycle, oncogenesis,
tional and physiological associations of SCFAs with liver apoptosis and proliferation (Brown et al., 2003; Natarajan and
diseases, general immunity, IBD, cardiovascular disease, and di- Pluznick, 2014; Smith et al., 2013). Moreover, dietary supply of
abetes were found in many epidemiological studies and in vari- SCFAs has recently been found to be able to protect against
ous in vivo and in vitro models (Corre ^a-Oliveira et al., 2016; type-I diabetes in mice model (Wen and Wong, 2017).
 460 | AFB1 DISRUPTS GUT-MICROBIAL METABOLISM

In addition to SCFAs, there are a great number of organic size (Rowland, 2012). In correspondence with the increase of
acids present in gut and feces that play important physiological cholic acid, we found severe liver damages and pathogenesis in
roles. They are either food-derived nutrients or the metabolic the AFB1-treated rats (Qian et al., 2013b, 2016). The abnormal re-
products generated in gut-microbiota and host metabolisms. duction of deoxycholic acid can be attributed to the relative
Interested organic acids in our study included fecal linoleic acid abundances of the deoxycholic acid-producing microbes, such
(cis-9, cis-12–18:2), pentadecanoic acid (15:0), pyruvic acid, 3- as Lachospiraceae, Clostridiaceae, and Ruminococcaceae, were all de-
phenyllactic acid, cholic acid, and deoxycholic acid, which were creased by AFB1 exposure (Wang et al., 2016). In both human
remarkably altered in the feces following AFB1 exposure studies and rodent models these strains can metabolize pri-
(Figure 4). Linoleic acid is an omega-6 polyunsaturated fatty mary bile acids into secondary bile acids (Labbe  et al., 2014).
acid known as an essential dietary nutrient that cannot be de There are also interactions among primary bile acids, secondary
novo synthesized by human body. The unsaturated fatty acids bile acids, and SCFAs in regulating host health, and the eleva-
are known to carry with various health-promoting functions, tion of intestinal primary bile acids with decreased secondary
such as antioxidant defense, suppression of blood levels of tri- bile acid was associated with the incidences of dysbiosis and
glycerides and cholesterol, maintenance of glucose tolerance, IBD in humans (Lefebvre et al., 2009). The increase of fecal cholic

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and mitigation of hyperinsulinemia (Whelan and Fritsche, acid in combination with the decrease of SCFAs were previously
2013). Most of these beneficial functions has been identified in observed in the patients with colon cancer (Weir et al., 2013).
conjugated linoleic acids, mainly as cis-9, trans-11 C18:2, trans-9, Pyruvic acid is a well-known energetic a-keto acid that is in-
trans-11 C18:2, and trans-10, cis-12 C18:2 (Worley and Powers, volved in a number of important metabolic pathways of both
2016; Yatsunenko et al., 2012). Pentadecanoic acid is known to gut-microbiota and host. It serves as energy supply to cells
carry a variety of regulatory functions in cell signaling, glucose through Krebs cycle, and can be transferred to SCFAs by
utilization, and the maintenance of the integrity and stability of Lactobacilli strains through glycolytic pathway (Pessione, 2012).
gut epithelium (Santaren et al., 2014). The abnormal accumula- Pyruvic acid can be transferred to carbohydrates via gluconeo-
tion of linoleic acid and pentadecanoic acid in rat feces sug- genesis, or participate in the biosynthesis of fatty acids after
gested a suppressed intestinal absorption of LCFAs, which is binding with acetyl-CoA (Kim et al., 2016). Since pyruvic acid
disadvantageous for host health. The deficient bioavailability takes such a central role in the catabolism of carbohydrates, its
may be caused by several conditions. First, the decrease of unusual accumulation in rat feces reflected a suppressed energy
SCFAs may affect the epithelial delivery of nutrients to hepatic utilization and disruption of glycolysis of gut-microbiota. This
portal vein, since SCFAs are well-known nutrients that are able may also result in the decrease of microbial synthesis of SCFAs
to enhance colonic blood flow and epithelial motility by provid- (VanHook, 2016). It seems that the reduction of SCFAs is not
ing energy and activating G-protein receptors (Scheppach, only caused by alteration of community structure of gut-
1994). Second, certain gut-microbial strains are capable of trans- microbiota, but also related with the specific metabolic path-
ferring LCFAs into their conjugated forms which are easier to be way. Last, 3-phenyllactic acid, a central intermediate product in
absorbed (Druart et al., 2014). For example, Lactobacillus, the upstream of phenylalanine catabolism (Stark et al., 1979),
Propionibacterium, and Bifidobacterium species can produce conju- was accumulated in the rat feces following exposure to AFB1.
gated linoleic acid from dietary linoleic acid by using microbial The abnormal accumulation of 3-phenyllactic acid suggested
lipoxygenases and cyclooxygenases—a process known to facili- the disruption of gut-microbial phenylalanine pathway
tate the absorption of LCFAs (Yatsunenko et al., 2012). Our previ- (Camilleri, 2011). The phenylalanine pathway is known to gen-
ous 16S rRNA analysis demonstrated that these strains were erate L-3, 4-dihydroxyphenylalanine (L-DOPA) and tyrosine.
suppressed by AFB1, which could affect the uptake and reduce L-DOPA is the precursor to a number of important neurotrans-
bioavailability of LCFAs (Wang et al., 2016). mitters such as dopamine, norepinephrine, and epinephrine. In
Bile acids are endogenous steroid acids synthesized from addition, L-DOPA itself also mediates neurotrophic factor re-
cholesterol by liver cells of most vertebrates. Different species lease by the brain and central neuro system (CNS) (Lopez et al.,
have distinct molecular forms of bile acids generated, but some 2008). For these reasons, the down-regulation of phenylalanine
major types of bile acids are shared by different species, eg, cho- pathway may interfere with host CNS function and cause-
lic acid and chenodeoxycholic acid in human and rat (Whittaker related health problems.
and Chipley, 1986). In human, bile acids are stored in the gall- Dietary AFB1 exposure and AFB1-induced adverse health
bladder, and are released into duodenum with bile juice under effects remain a major public health problem in many tropical de-
the dietary stimulation. Upon arriving small intestine, bile acids veloping nations. The range of dosage used in this study (5–75 mg/
participate in the digestion and absorption of fats and fat- kg b.w.) was relevant to human exposure, based on 300 g corn con-
soluble vitamins and can be further metabolized into a variety sumption per day (Gwirtz and Garcia-Casal, 2014) and oral expo-
of secondary metabolites by gut-microbiota. In this work, cholic sure levels ranged from 100 to 1000 lg/kg corn for high-risk human
acid and deoxycholic acid were selected as representative pri- populations in Kenya, Ghana, and Guangxi area of China (Azziz-
mary and secondary bile acids to probe the microbial metabo- Baumgartner et al., 2005; Groopman et al., 1992; Tang et al., 2009).
lism of bile acids, since they are found in both human and rat The dose was multiplied by an adjusting factor of 6.2 in order to
feces at comparatively high levels. We found a remarkable ele- transfer human exposure to that in rats (Nair and Jacob, 2016).
vation of cholic acid level with a significant reduction of deoxy- Regarding the mechanisms behind the metabolite altera-
cholic acid level in AFB1 exposed rat feces. The significant tions found in this study, there are several mechanisms in-
elevation of cholic acid is generally considered to be harmful to volved: (1) AFB1, as a natural antimicrobial agent, can
host health. Abnormal increase of cholic acid is associated with selectively inhibit certain bacterial strains and influence on the
liver pathogenesis such as cirrhosis and steatosis (Mouzaki growth of other strains (Arai et al., 1967; Haskard et al., 2001), as
et al., 2016), and is also known as a risk factor for intestinal in- shown in the compositional changes of gut-microbiota revealed
flammation (Camilleri, 2011). Besides, extra cholic acid in gut by 16 s rRNA analysis; (2) AFB1, as a potent hepatic toxin, can
may partially contribute to the incidence of colon cancer by damage liver—the major metabolic organ and in turn induce
stimulating the growth of a small-size benign adenoma to larger the metabolic changes for the supply of nutrients and
 ZHOU ET AL. | 461

F344 male rats treated with repeated-dose 5–75 μg AFB1 /kg B.W.

Biochemical, Growth, & 16s rRNA Sequencing

Enterohepac
Histopathological Alteraons Clostridiales Roseburia
Cross-talk
2-wk
Lactobacillales Streptococcus
Serum AFB-Lys ↑; GST-P+ foci ↑; B. W. ↓
Gut Epithelium
3-wk Heptac Portal Vein Clostridiales spp.
Serum AFB-Lys ↓; GST-P+ foci ↑; B. W.
Alteraons in Microbial Metabolites
↓; Necrosis ↑; Bile duct proliferaon ↑;
B. W. ↓ Pyruvic acid & 3-Phenyllacc acid
4-wk Long chain fay acids
Serum AFB-Lys ↓; GST-P+ foci ↑; B. W. Cholic acid
↓; Necrosis ↑; Bile duct proliferaon ↑;
Short chain fay acids
Clear cell foci ↑; B. W. ↓

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Deoxycholic acid
Bile Duct
Liver Gut-microbiota
Altered immune funcons
Inflammatory factors Stunted growth
Metabolic syndromes
Heptac Artery Nutrional deficiency
Suscepble to infecous diseases
Liver cancer Adverse Health Effects
Figure 5. Summary of the adverse health outcomes associated with dietary exposure to AFB1 in F344 rat model. Gray arrow indicates the changing trends of microbial taxa,
biomarkers, phenotypes, and metabolites induced by AFB1-treatment. The establishment of rat model for AFB1 oral exposure, as well as the 16s rRNA analysis have been
published already (Qian et al., 2013a,b, 2014; Wang et al., 2016). Briefly, male F344 rats were gavaged with AFB1 at doses of 0, 5, 10, 25, 50, and 75 lg/kg b.w. per day. The major
pathological changes are summarized on the left panel. After 3 weeks of exposure to 75 lg AFB1/kg b.w., bile duct proliferation, liver GST-Pþ foci co-occurred, followed by pro-
liferation foci formation after 4 weeks and dramatic alanine transaminase, aspartate transaminase and creatine kinase elevations after 5 weeks of treatment.

metabolites to host cells and tissues, including gut cells, which Program at the University of Georgia Graduate School pro-
may play an important role in the metabolism of gut- vided stipend supports.
microbiota (Atroshi et al., 1998). However, the more specific
mechanism related to how AFB1 induces changes of gut-
microbiota community structure and the dependent metabo-
FUNDING
lites still need to be clarified in future study. This work was supported partially by the research contract
Taken together, as summarized in Figure 5 based on our pre- (ECG-A-00-07-00001-00), from the United States Agency for
vious studies (Qian et al., 2013a,b, 2014; Wang et al., 2016), oral ex- International Development via Peanut CRSP and the Center
posure to AFB1 in rat results in significant toxic effects, for Mycotoxin Research at the College of Public Health,
biochemical alterations, and induction of preneoplastic GST-P
University of Georgia.
positive liver foci. With same study design, here we show that
AFB1 can induce the adverse change of community structure of
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