toxins
Article
Aflatoxin B1 Exposure in Sheep: Insights into Hepatotoxicity
Based on Oxidative Stress, Inflammatory Injury, Apoptosis, and
Gut Microbiota Analysis
Yuzhen Sui , Ying Lu, Shoujun Zuo, Haidong Wang, Xiaokun Bian, Guizhen Chen, Shucheng Huang
Hongyu Dai, Fang Liu * and Haiju Dong *
Citation: Sui, Y.; Lu, Y.; Zuo, S.;
Wang, H.; Bian, X.; Chen, G.; Huang,
S.; Dai, H.; Liu, F.; Dong, H. Aflatoxin
B1 Exposure in Sheep: Insights into
Hepatotoxicity Based on Oxidative
Stress, Inflammatory Injury,
Apoptosis, and Gut Microbiota
Analysis. Toxins 2022, 14, 840.
https://doi.org/10.3390/
toxins14120840
Received: 5 November 2022
Accepted: 29 November 2022
Published: 1 December 2022
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Attribution (CC BY) license (https://
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4.0/).
Toxins 2022, 14, 840. https://doi.org/10.3390/toxins14120840
,
International Joint Research Center of National Animal Immunology, College of Veterinary Medicine,
Henan Agricultural University, Zhengzhou 450046, China
* Correspondence: liufang.vet@henau.edu.cn (F.L.); dongju0528@163.com (H.D.)
Abstract: The widespread fungal toxin Aflatoxin B1 (AFB1 ) is an inevitable pollutant affecting the
health of humans, poultry, and livestock. Although studies indicate that AFB1 is hepatotoxic, there
are few studies on AFB1 -induced hepatotoxicity in sheep. Thus, this study examined how AFB1
affected sheep liver function 24 h after the animals received 1 mg/kg bw of AFB1 orally (dissolved
in 20 mL, 4% v/v ethanol). The acute AFB1 poisoning caused histopathological injuries to the liver
and increased total bilirubin (TBIL) and alkaline phosphatase (AKP) levels. AFB1 also markedly
elevated the levels of the pro-inflammatory cytokines TNF-α and IL-6 while considerably reducing
the expression of antioxidation-related genes (SOD-1 and SOD-2) and the anti-inflammatory gene
IL-10 in the liver. Additionally, it caused apoptosis by dramatically altering the expression of genes
associated with apoptosis including Bax, Caspase-3, and Bcl-2/Bax. Notably, AFB1 exposure altered the
gut microbiota composition, mainly manifested by BF311 spp. and Alistipes spp. abundance, which
are associated with liver injury. In conclusion, AFB1 can cause liver injury and liver dysfunction in
sheep via oxidative stress, inflammation, apoptosis, and gut-microbiota disturbance.
Keywords: Aflatoxin B1 ; liver function; oxidative stress; inflammatory; apoptosis; gut microbiota
Key Contribution: Our experiments confirmed that AFB1 can cause liver injury and liver dysfunction
in sheep through oxidative stress, inflammation, apoptosis, and gut-microbiota disturbance.
1. Introduction
Mycotoxins are widespread in all processes of agricultural production and can se-
riously endanger food and feed safety, threatening human and animal health [1]. Over
4.5 billion people are at high risk of exposure to these food contaminants, and it causes
annual economic losses of hundreds of billions of dollars. According to the Food and
Agriculture Organization of the United Nations (FAO), about 25% of the world’s crops
are polluted by mycotoxins in varying degrees [2,3]. Aflatoxin B1 (AFB1 ), the most toxic
mycotoxin, is hepatotoxic, teratogenic, and mutagenic, and it is classified as a class I car-
cinogen by the World Health Organization [4,5]. The sensitivity of different livestock
species to AFB1 varies [6,7]. Chickens are highly sensitive to AFB1 , and poisoning is
caused mostly by long-term consumption of AFB1 -contaminated feed [5,7]. Although
sheep are typically thought to have significant tolerance to AFB1 due to their status as
ruminants, poisoning can, nonetheless, occur from prolonged or excessive consumption of
an AFB1 -containing diet [8].
As a hepatotoxic chemical substance, AFB1 is activated into AFB1 epoxide under the
action of the cytochrome P450 (CYP450) enzyme, inducing liver cancer and hepatotoxic-
ity [9]. AFB1 toxicity can lead to oxidative damage, apoptosis, and inflammation, resulting
in liver congestion, pale coloration, enlargement, and necrosis, inducing chronic and acute
https://www.mdpi.com/journal/toxins
Toxins 2022, 14, 840 2 of 14

hepatocellular injury [7,10–12]. Livestock and poultry can accumulate a certain level of
AFB1 through dietary exposure, leading to chronic and acute liver diseases in humans via
biological accumulation in the food chain [13].
The liver is an important detoxification center as well as a target organ for the metabolic
transformation of AFB1 in the body, and it is crucial for the body’s defense against xeno-
biotics [14]. Past studies on the hepatotoxic effects of AFB1 exposure in sheep have not
explored the association between changes in liver function indicators and oxidative stress,
inflammation, apoptosis, or gut microbiota. Therefore, we sought to assess the liver func-
tion, oxidation, inflammation, and apoptosis indices and composition of the fecal microbial
community to offer theoretical support and experimental data on liver damage caused by
AFB1 exposure in sheep.

2. Results
2.1. Effects of AFB1 Exposure on Clinical Symptoms, Body Temperature, Respiration, Heart Rate,
and Conjunctival Color
Sheep in the AFB1 group showed poor mental status, foaming at the mouth, and re-
duced food intake after gavage compared to the control group. Before AFB1 exposure, there
were no significant differences between the control and AFB1 group body temperatures,
respiration, or heart rates, as indicated in Table S2 (p = 0.280, 0.280, and 0.146, respectively).
Nevertheless, 24 h after AFB1 exposure, the respiration and heart rate of the AFB1 group
were lower than those of the control group (p = 0.071 and 0.084, respectively), while the
body temperature was higher (p = 0.519). The AFB1 group had lower changes (slope)
in respiration and heart rate than the control group (p = 0.488 and 0.022, respectively).
Furthermore, the temperature change in the AFB1 group was greater than in the control
group (p = 0.043).
As can be observed in Figure S1A–C, the conjunctival color L of the AFB1 group
dropped considerably (p = 0.005) after acute exposure compared with the control group,
although redness (a value) and yellowness (b value) did not differ significantly (p = 0.442
and 0.262, respectively).

2.2. Effects of AFB1 Exposure on Serum Biochemical Indices

As shown in Table S3, serum calcium (Ca) levels in the AFB1 group were considerably
lower (p = 0.006) than those in the control group, while serum total protein (TP), albumin
(ALB), globulin (GLO), white sphere ratio (A/G), amylase (AMY), phosphorus (P), and
glucose (GLU) levels showed insignificant decreasing trends (p > 0.05). Furthermore,
serum total bilirubin (TBIL) levels were considerably higher (p = 0.002), and serum alanine
aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), urea nitrogen
(BUN), BUN/CRE, and creatine kinase (CK) levels increased in the AFB1 group compared
with the control group (p = 0.297, 0.190, 0.306, 0.599, 0.938, and 0.41, respectively). The
increased serum indices of AST, ALT, and TBIL suggests that AFB1 exposure could result
in liver dysfunction (Table S3).

2.3. Effects of AFB1 Exposure on Liver Tissue Structure and Liver Tissue Function
As shown in Figure 1A–C, the ALT, AST, and AKP levels in the AFB1 group increased
when compared with those of the control group. Among them, the AKP level was consider-
ably higher than that in the control group (p = 0.007).
Figure 1D–G depicts liver tissue stained with H&E. The liver lobules of the control
group had normal shape; the cords were arranged in an orderly, radial pattern; and the liver
cells were complete and uniform in size and cytoplasm. The liver tissue of the AFB1 group,
in contrast, exhibited pathological changes, including abnormal hepatic lobule structure,
disorganized hepatic cord arrangement, swollen and unevenly sized hepatocytes, severe
infiltration of inflammatory cells, vacuolar degeneration, and necrosis in some hepatocytes.
 group, in contrast, exhibited pathological changes, including abnormal hepatic lobule
structure, disorganized hepatic cord arrangement, swollen and unevenly sized hepato-
Toxins 2022, 14, 840 cytes, severe infiltration of inflammatory cells, vacuolar degeneration, and necrosis in
3 of 14
some hepatocytes.

Figure 1. Effects of AFB1 exposure on liver function and morphological structure of liver tissue.
(A–C) Liver function indexes of liver tissue (ALT, AST, and AKP). (D,E) H&E staining reveals normal
Figure 1. Effects of AFB1 exposure on liver function and morphological structure of liver tissue. (A–
liver tissues.
C) Liver (F,G) indexes
function Liver morphology after(ALT,
of liver tissue AFB1AST,
exposure. Note (D,E)
and AKP). the presence of ruptured
H&E staining cells
reveals and
normal
inflammatory cell infiltration (arrow). 100 µm (D,F), 50 µm (E,G). ** p < 0.01.
liver tissues. (F,G) Liver morphology after AFB1 exposure. Note the presence of ruptured cells and
inflammatory cell infiltration (arrow). 100 μm (D,F), 50 μm (E,G). ** p < 0.01.
2.4. Effects of AFB1 Exposure on Liver Oxidative Damage
A decrease
2.4. Effects of AFBin1 Exposure
liver function is related
on Liver Oxidativeto Damage
oxidative stress [15]. Measurements were
taken of the concentrations of MDA and T-AOC and the activities of antioxidant enzymes
A decrease in liver function is related to oxidative stress [15]. Measurements were
(CAT, SOD). There was no significant difference in MDA, SOD, CAT, and T-AOC levels
taken of the concentrations of MDA and T-AOC and the activities of antioxidant enzymes
between the two groups (p > 0.05; Figure 2A–D). Notably, MDA concentration and SOD
(CAT, SOD). There was no significant difference in MDA, SOD, CAT, and T-AOC levels
activity showed an increasing and decreasing trend compared with the control group,
between the two groups (p > 0.05; Figure 2A–D). Notably, MDA concentration and SOD
respectively. Protein and mRNA levels typically show reasonable correlation [16], so we
activity showed an increasing and decreasing trend compared with the control group,
estimated the expression of SOD-1 and SOD-2 genes in liver tissues. SOD-1 and SOD-2
respectively.
levels in the AFBProtein and mRNA levels typically show reasonable correlation [16], so we
1 group were considerably lower than those in the control group (p < 0.001;
estimated
Figure 2E,F). the expression of SOD-1 and SOD-2 genes in liver tissues. SOD-1 and SOD-2
levels in the AFB 1 group were considerably lower than those in the control group (p <
The relationship between antioxidant indexes and liver function-related indexes was
0.001; Figureusing
investigated 2E,F).Pearson correlation analysis (Figure 2G). The levels of AKP (tissue) were
The relationship
negatively correlated with between
thoseantioxidant indexesand
of T-AOC, SOD-1, andSOD-2
liver function-related indexes
(r = −0.639, −0.175, was
−0.411,
investigated
and p = 0.025,using
0.03, Pearson correlation
and 0.002, analysis
respectively). (Figure
SOD-1 (r = 2G). The plevels
−0.893, of AKP
= 0.001) (tissue)
and SOD-2
were negatively correlated with those of T-AOC, SOD-1, and SOD-2
(r = −0.760, p = 0.029) levels were also negatively correlated with TBIL (serum).(r = −0.639, −0.175,
−0.411, and p = 0.025, 0.03, and 0.002, respectively). SOD-1 (r = −0.893, p = 0.001) and SOD-
2 (r = −0.760, p = 0.029) levels were also negatively correlated with TBIL (serum).
Toxins 2022, 14, x FOR PEER REVIEW 4 of 16

Toxins 2022, 14, x FOR PEER REVIEW 4 of 16

Toxins 2022, 14, 840 4 of 14

Figure 2. Effects of AFB1 exposure on liver oxidative damage in sheep. (A–D) Liver tissue MDA,
CAT, SOD, and T-AOC levels. (E,F) Relative expressions of the genes SOD-1, and SOD-2 related to
Figure 2. Effects of AFB1 exposure on liver oxidative damage in sheep. (A–D) Liver tissue MDA,
oxidative stress in the liver. (G) Pearson correlation analysis of oxidative stress indexes and param-
CAT, SOD, and T-AOC levels. (E,F) Relative expressions of the genes SOD-1, and SOD-2 related
eters related to liver function. * p < 0.05, ** p < 0.01, *** p < 0.001.
to oxidative
Figure stress
2. Effects ofin
AFBthe1 exposure
liver. (G)on Pearson correlation
liver oxidative analysis
damage of oxidative
in sheep. (A–D) stress indexes
Liver tissue and
MDA,
CAT, SOD,ofrelated
parameters
2.5. Effects and
AFB T-AOC levels.
to liver (E,F) *Relative
function. p < 0.05,expressions
** p < 0.01, of***the
p <genes
0.001.SOD-1, and SOD-2 related to
1 Exposure on the Expression of Inflammation-Related Factors in the Liver
oxidative stress in the liver. (G) Pearson correlation analysis of oxidative stress indexes and param-
etersTo
2.5. further
Effects
relatedoftoAFBinvestigate
liver function.the
1 Exposure *on effects
p <the ** of
p <AFB
Expression
0.05, onp liver
of1 ***
0.01, inflammation,
Inflammation-Related
< 0.001. we examined
Factors in the Liverthe
expression of theinvestigate
To further inflammatory the cytokines
effects of TNF-α,
AFB1 onIL-6, liverand IL-1β and anti-inflammatory
inflammation, we examined the
cytokine IL-10 in
2.5. Effects ofofAFB
expression the liver tissues
Exposure on the
the 1inflammatory of sheep
Expression
cytokines in each group (Figure
of Inflammation-Related
TNF-α, IL-6, and IL-1β 3A–D). Theinresults
Factors
and indi-
the Liver
anti-inflammatory
cate that
cytokine the AFB group
IL-10 ininvestigate
To further 1 had
the liver tissuessignificantly
of sheep
the effects higher
ofinAFB
each levels
group
1 on
of IL-1β
liver(Figure and IL-6
3A–D). The
inflammation, expression
weresults than
indicate
examined the
those
that of
the the
AFB control
group group
had (p = 0.021
significantly and 0.003,
higher respectively).
levels of IL-1β Furthermore,
and
expression of the inflammatory cytokines TNF-α, IL-6, and IL-1β and anti-inflammatory
1 IL-6 IL-10
expression expres-
than those
sion
of wascontrol
the
cytokine considerably
in the lower
IL-10group liver in the
(p = 0.021
tissues AFB
and 1 group
0.003,
of sheep (p = 0.034),
groupalthough
respectively).
in each TNF-αIL-10
Furthermore,
(Figure 3A–D). expression
The did
expression
results indi-
not
was change significantly
considerably lower (p
in = 0.641).
the
cate that the AFB1 group had significantly AFB Pearson
1 group correlation
(p = 0.034), analysis
although revealed
TNF-α that IL-6
expression
higher levels of IL-1β and IL-6 expression than gene
did
expression
not change was positively
significantly correlated
(p = 0.641). to serum
Pearson TBIL levels
correlation (r = 0.798
analysis
those of the control group (p = 0.021 and 0.003, respectively). Furthermore, IL-10 expres- and p
revealed = 0.01;
that Figure
IL-6 gene
3E).
expression was positively
sion was considerably correlated
lower to serum
in the AFB TBIL levels (r = 0.798 and p = 0.01; Figure 3E).
1 group (p = 0.034), although TNF-α expression did

not change significantly (p = 0.641). Pearson correlation analysis revealed that IL-6 gene
expression was positively correlated to serum TBIL levels (r = 0.798 and p = 0.01; Figure
3E).

Figure 3. Effects of AFB1 exposure on inflammation-related indexes in sheep. (A–D) TNF-α,

IL-1β, IL-6, and IL-10 mRNA levels detected in liver. (E) Heat map showing the correlation between
indicators of liver function and levels of inflammatory gene expression. * p < 0.05, ** p < 0.01.
 IL-6, and IL-10 mRNA levels detected in liver. (E) Heat map showing the correlation between indi-
cators of liver function and levels of inflammatory gene expression. * p < 0.05, ** p < 0.01.

2.6. Effects of AFB1 Exposure on Liver Cell Apoptosis and Apoptosis-Related Gene Expression.
TUNEL staining in the liver of sheep in each group is shown in Figure 4A,B. The
Toxins 2022, 14, 840
AFB1 group had a considerably higher positive rate of TUNEL than the control group.5 We of 14

also detected the mRNA expression of Caspase-3, Bax, and Bcl-2 in the hepatocytes. As
seen in Figure 4C,D, the Bcl-2/Bax ratio was reduced dramatically (p < 0.001) in the AFB1
2.6. Effects
group of AFB1with
compared Exposure on Liver
the control Cell Apoptosis
group, and Apoptosis-Related
whereas Caspase-3 Geneincreased
gene expression Expressionsig-
nificantly (p = 0.002).
TUNEL staining in the liver of sheep in each group is shown in Figure 4A,B. The AFB1
groupAccording to the Pearson
had a considerably highercorrelation
positive analysis, AKP (tissue)
rate of TUNEL wascontrol
than the negatively correlated
group. We also
with the Bcl-2/Bax ratio (r = −0.762 and p = 0.004) and positively
detected the mRNA expression of Caspase-3, Bax, and Bcl-2 in the hepatocytes. As correlated with the
seen in
Caspase-3 gene (r = 0.730 and p = 0.07; Figure 4E). The Bcl-2/Bax ratio and serum
Figure 4C,D, the Bcl-2/Bax ratio was reduced dramatically (p < 0.001) in the AFB1 group TBIL level
had a negative
compared correlation
with the (r = −0.883
control group, and Caspase-3
whereas p = 0.002; gene
Figure 4E).
expression increased significantly
(p = 0.002).

Figure 4. Effects of AFB1 exposure on hepatocyte apoptosis indexes in sheep. (A) TUNEL staining of
sheep liver tissue, with green fluorescence indicating TUNEL positive cells and DAPI staining for
Figure 4. Effects of AFB1 exposure on hepatocyte apoptosis indexes in sheep. (A) TUNEL staining
nucleus (400×). (B) TUNEL-positive cell percentage. (C) Bcl-2 to Bax ratio. (D) Caspase 3 mRNA
of sheep liver tissue, with green fluorescence indicating TUNEL positive cells and DAPI staining for
level detected
nucleus in (B)
(400×). liverTUNEL-positive
using RT-qPCR.cell
(E)percentage.
Heat map showing
(C) Bcl-2the
to correlation between
Bax ratio. (D) indicators
Caspase 3 mRNA of
liver function and hepatocyte apoptosis indexes. * p < 0.05, ** p < 0.01, *** p < 0.001.
level detected in liver using RT-qPCR. (E) Heat map showing the correlation between indicators of
liver function and hepatocyte apoptosis indexes. * p < 0.05, ** p < 0.01, *** p < 0.001.
According to the Pearson correlation analysis, AKP (tissue) was negatively correlated
with the Bcl-2/Bax
2.7. Changes in Gut Microbiota −0.762 by
ratio (r = Induced and p 1=Exposure
AFB 0.004) and positively correlated with the
Caspase-3 gene (r = 0.730 and p = 0.07; Figure 4E). The Bcl-2/Bax ratio and serum TBIL level
An increasing number of studies show that changes in gut microbiota composition
had a negative correlation (r = −0.883 and p = 0.002; Figure 4E).
and function are crucial for liver health [17]. To study how the composition of gut micro-
biota
2.7. changed
Changes afterMicrobiota
in Gut AFB1 exposure,
Induced16S
byrRNA gene sequencing was performed. The AFB1
AFB1 Exposure
and control groups had coverage rates of 98.39% and 96.46%, respectively, indicating that
An increasing number of studies show that changes in gut microbiota composition and
the majority of the gut microbiota diversity was detected (Figure 5A). In comparison to
function are crucial for liver health [17]. To study how the composition of gut microbiota
the control group, the observed species, Shannon, Simpson, and Good’s coverage indexes
changed after AFB1 exposure, 16S rRNA gene sequencing was performed. The AFB1 and
in the AFB1 group were reduced (p = 0.39, 0.021, 0.021, and 0.25, respectively; Figure 5A).
control groups had coverage rates of 98.39% and 96.46%, respectively, indicating that the
The gut microbiota composition showed a trend of relative separation (p = 0.06) for Beta
majority of the gut microbiota diversity was detected (Figure 5A). In comparison to the
control group, the observed species, Shannon, Simpson, and Good’s coverage indexes in the
AFB1 group were reduced (p = 0.39, 0.021, 0.021, and 0.25, respectively; Figure 5A). The gut
microbiota composition showed a trend of relative separation (p = 0.06) for Beta diversity
between the control and AFB1 groups (PCo1 contribution of 27.80%, PCo2 contribution
of 22.80%; Figure 5B). A total of 2204 ASVs were found in both groups, with 9768 and
6632 ASVs found in the control and AFB1 groups, respectively (Figure 5C). Firmicutes,
Bacteroidetes, Spirochaetes, Verrucomicrobia, and Proteobacteria were the top five abundant
phyla in both groups, as shown in Figure 5D. Firmicutes, Spirochaetes, Verrucomicrobia, and
Proteobacteria were more abundant in the AFB1 group (73.49%, 0.96%, 0.99%, and 0.61%,
respectively) than in the control group (72.23%, 0.56%, 0.49%, and 0.56%, respectively),
Toxins 2022, 14, 840 6 of 14

while Bacteroidetes were more abundant in the control group (24.32%) than in the AFB1
group (23.01%; Figure 5D–I). The Firmicutes/Bacteroides ratio (F/B ratio) was higher in the
AFB1 group than in the control group, but the difference was not significant (p = 0.564;
Figure 5J).
The top 30 genera were also identified. Among them, oscillospira spp., 5-7N15 spp.,
and (Clostridium) spp. were the most predominant in the control group, and oscillospira spp.,
5-7N15 spp., and BF311 spp. accounted for the predominance in the AFB1 group (Figure 5K).
LEfSe analysis was utilized to further detect the differential bacterial taxa between the
two groups, and 33 discriminative features were identified (genus level; LDA score > 2,
p < 0.05; Figure 5L). Among them, BF311 spp., Flavobacterium spp., and (Clostridium) spp.
were concentrated in the AFB1 group, while Ruminococcaceae spp., Alistipes spp., and
Roseburia spp. were concentrated in the control group (Figure 5L,M).
The top 30 most important genera were screened using random forest analysis,
and the most important species at the genus level were BF311 spp., Roseburia spp., and
Butyrivibrio spp. (Figure 6A). Thirty-three characteristic species were identified using a
Venn diagram analysis of the LEfSe results (LDA score > 2; p < 0.05), and 10 genera were
screened out from the top 30 genera based on significance scores (Figure 6B).
Furthermore, we noted that the abundances of BF311 spp. and (Clostridium) spp. were
higher in the AFB1 group, whereas those of Alistipes spp., Roseburia spp., Mogibacterium spp.,
Butyrivibrio spp., rc4-4 spp., Coprococcus spp., L7A_E11 spp., and Paraprevotella spp. were
lower in the AFB1 group than in the control group (p = 0.155, 0.074, 0.145, 0.108, 0.102, 0.093,
0.015, 0.037, 0.022, and 0.066 respectively; Figure 6C). Pearson correlation analysis revealed
that the TBIL (serum) level was significantly negatively correlated with Alistipes spp.
Toxins 2022, 14, x FOR PEER REVIEW
(r = 0.84, p = 0.036; Figure 6D), whereas AKP (tissue) levels were positively correlated7 with
of 16

BF311 spp. (r = 0.897, p = 0.002; Figure 6D).

Figure 5. Cont.
Toxins 2022, 14, 840 7 of 14

Figure 5. Effects of AFB1 exposure on gut microbiota structure. (A) Alpha diversity indexes (observed
species, Shannon, Simpson, and Good’s coverage) of intestinal microbial. (B) Principal Coordination
Analysis (PCoA) of the intestinal microbiome in the AFB1 and control groups (n = 4). (C) ASVs
Venn diagram. (D) The top five phylum-level abundances of gut microbiota. (E–J) Difference in the
abundances of gut microbiota (Firmicutes, Bacteroidetes, Spirochaetes, Verrucomicrobia, Proteobacteria,
and F/B ratio, respectively) at the phylum level. (K) Gut microbiota relative abundance at the genus
level (Top 30). (L,M) LDA score and cladogram of LDA effect size (LEfSe). * p < 0.05.
Toxins 2022,14,
Toxins2022, 14,840
x FOR PEER REVIEW 98 of 14
16

Figure 6. Analysis of differential gut microbiota. (A) Gut microbiota random forest analysis at the
Figure 6. Analysis of differential gut microbiota. (A) Gut microbiota random forest analysis at the
genera level (Top 30). (B) Venn diagrams for the LEfSe and the random forest. (C) The analysis
genera level (Top 30). (B) Venn diagrams for the LEfSe and the random forest. (C) The analysis of
of relative abundances of the ten genera between groups. (D) Heat map showing the correlation
relative abundances of the ten genera between groups. (D) Heat map showing the correlation be-
between indicatorsofofliver
tween indicators liverfunction
functionand
andgut microbiota.* p* p< <
gutmicrobiota. 0.05,
0.05, p <p 0.01,
** ** < 0.01, p <p 0.001.
****** < 0.001.

3. Discussion
3. Discussion
Body temperature, breathing rate, and heart rate are important indicators of physical
healthBody
[18].temperature,
In this study,breathing
the bodyrate, and heart of
temperatures rate are important
sheep increasedindicators of physical
and respiration and
health [18]. In this study, the body temperatures of sheep increased and
heart rates decreased after intake of AFB1 . AFB1 exposure-induced gastrointestinal and respiration and
heart rates decreased after intake of AFB 1. AFB1 exposure-induced gastrointestinal and
neurological symptoms were also observed in this study. These results suggest that AFB1
neurological
exposure symptoms
affects the healthwere also observed in this study. These results suggest that AFB1
of sheep.
exposure
Jaundice is an importantsheep.
affects the health of sign of liver dysfunction [19]. As AFB1 targets the liver,
Jaundice
exposure to AFBis anmay
important
cause sign of liver
jaundice dysfunction
and change [19].of
the color Asthe
AFB 1 targets the liver, ex-
conjunctiva. Therefore,
1
posure to AFB 1 may cause jaundice and change the color of the conjunctiva. Therefore, we
we measured conjunctival color and noted that AFB1 exposure significantly reduced the
Lmeasured conjunctival
values. However, color and difference
no significant noted thatinAFB 1 exposure
b and a valuessignificantly
was detectedreduced
betweenthetheL
values. However, no significant difference in b and a values was detected
two groups, which could be attributed to the short-term exposure of the animals to AFB between the
1
two groups,
in this study. which could be attributed to the short-term exposure of the animals to AFB 1

in this study.
Toxins 2022, 14, 840 9 of 14

The activities of serum enzymes such as ALT and AST, as well as TP, ALB, TBIL, and GLO
concentration, have been defined as critical indicators of liver injury and function [20–22]. In
this investigation, AFB1 exposure significantly increased the serum TBIL content of sheep,
while the levels of serum TP, ALB, GLO, and A/G tended to decrease, and AST and ALT
contents tended to increase. These phenomena suggest that exposure to AFB1 may harm
the liver and impair liver function in sheep [23]. BUN and CRE are the main biochemical
indexes of renal function, which increase with the aggravation of renal injury [24]. In this
study, BUN and CRE levels tended to increase slightly, which may have been caused by the
short acting time of AFB1 in this study. Ca2+ is an important signaling molecule involved
in numerous cellular processes, and its content decreases with the aggravation of liver and
kidney diseases [25]. AFB1 exposure significantly decreased the serum Ca content in sheep,
indicating the possibility of liver damage. We next assessed liver tissue to explore whether
AFB1 exposure would negatively affect sheep liver tissue.
AFB1 exposure resulted in hepatocyte degeneration and considerable inflammatory
cell infiltration, suggesting liver tissue damage and corroborating the results of Tsiouris et al.
(2021) [26]. In general, elevated ALT, AST, and AKP levels indicate liver dysfunction, which
is critical for the differential diagnosis of liver diseases [27]. In the present study, AFB1
exposure increased ALT, AST, and AKP levels in the liver tissue of sheep compared with
those of the control group, reflecting the negative impact of AFB1 exposure on liver function.
Numerous studies have demonstrated that AFB1 increases lipid peroxidation and
induces the formation of high levels of reactive oxygen species and free radicals, dam-
aging body organs through oxidative stress [28,29]. Since MDA is a byproduct of lipid
peroxidation, the level of lipid peroxidation can be inferred from the MDA content in liv-
ing things [30,31]. In this study, the MDA content increased slightly, indicating liver
tissue injury. Antioxidant markers, including SOD, CAT, and T-AOC, are known to
play key roles in attenuating oxidative stress by scavenging reactive oxygen species [32].
Although SOD and T-AOC levels were both reduced in the liver of sheep exposed to AFB1
compared to those of the control group, the difference was not significant. The activity
of SOD, a particular antioxidant enzyme that neutralizes superoxide anions in ROS free
radicals, indirectly reflects the capacity to neutralize oxygen free radicals and is crucial in
liver injury [33]. Further examination of gene expression revealed that SOD-1 and SOD-2
levels were significantly decreased. However, an apparent increase in the CAT content
was observed in the present study, which may have resulted from the defense mechanism
of the body against AFB1 toxicity [8,34]. In this context, Cao et al. (2021) discovered that
AFB1 intoxication significantly increases CAT activity in sheep exposed with AFB1 [8]. In
addition, the correlation between liver function and antioxidant enzyme activity revealed
that AFB1 exposure causes a decrease in liver function related to oxidative damage.
The inflammatory response is an important mechanism in AFB1 toxicity [35]. Ox-
idative stress can increase the production of different types of ROS in the body, and ROS
can activate the nuclear factor-kappa B (NF-κB) pathway, leading to the production of
inflammatory cytokines [36]. TNF-α is the most prominent pro-inflammatory cytokine
involved in the activation of NF-κB, which induces the expression of IL-1β, IL-6, and other
downstream inflammatory mediators [37]. AFB1 significantly increased the expression
of the inflammatory factors IL-1β and IL-6, according to our findings. Furthermore, the
anti-inflammatory IL-10 gene, which is linked to liver function, was drastically downregu-
lated. The changes in the inflammatory factor expression levels in the liver tissue imply
that exposure to AFB1 causes inflammatory injury to the liver.
It has been reported that AFB1 destroys the integrity of the cell membrane by stimu-
lating phospholipids and inducing ROS formation [38]. When excessive ROS is produced
and the scavenging capacity of the body decreases, it can lead to protein, DNA, and mi-
tochondrial damage, thus inducing apoptosis [39]. Excessive apoptosis can lead to organ
damage, which is considered one of the mechanisms of AFB1 -induced toxicity [40]. Our
findings showed that when compared with the control group, the AFB1 group had a sig-
nificantly higher rate of hepatocyte apoptosis detected using the TUNEL method. These
Toxins 2022, 14, 840 10 of 14

results are similar with the findings of a prior study by Xu et al. (2021) [41]. The release of
mitochondrial cytochrome c is affected by changes in the ratio of Bcl-2 to Bax expression in
cells, and a decrease in this ratio results in apoptosis, according to studies conducted on
mammals [42,43]. At the same time, caspase-3 is a common effector of apoptosis [44]. In
this study, we discovered that after AFB1 exposure, Bcl-2/Bax gene expression decreased
while caspase-3 gene expression increased, indicating that AFB1 exposure can promote liver
cell apoptosis. In addition, the correlation between liver function and apoptosis showed
that the decline in liver function caused by AFB1 exposure is related to apoptosis.
Like most mycotoxins, AFB1 not only directly damages body organs but also interferes
with the normal activities of animal intestinal flora via enterohepatic circulation [7,8]. For
example, long-term feeding of AFB1 can significantly reduce most intestinal microbiota in
mice [45], and microbiota decline was observed in the acute AFB1 poisoning experiment in
this study. Moreover, intestinal flora can combine, transform, degrade, and transfer myco-
toxins; promote the healthy growth of livestock and poultry; and participate in material
metabolism [46–48]. Through amplicon sequencing, we discovered that Firmicutes and
Bacteroidetes were the two largest phyla that made up the sheep intestinal flora, which is
consistent with earlier research on mammalian intestinal flora [49]. Firmicutes/Bacteroidetes
ratios are typically correlated with inflammatory marker levels and pathological conditions
of intestinal metabolic homeostasis [49]. AFB1 exposure significantly raised the F/B ratio
in this study, implying that AFB1 exposure may disrupt gut metabolic homeostasis and
alter inflammatory metabolite levels. At the same time, by screening at the genus level,
we identified two genera that deserve attention: BF311 spp. and Alistipes spp. Although
there are few studies on the function of BF311 spp., some suggest that BF311 spp. may
play a crucial role in the rumen ecosystem and even in rumen synchronization [50]. As a
relatively newly identified bacterial genus, Alistipes spp. has been shown to be associated
with liver fibrosis, cardiovascular disease, cancer immunotherapy, cardiovascular disease,
colitis, and depression [51]. The decrease of Alistipes spp. causes a reduction in short-chain
fatty acids (SCFA), which further leads to a decrease in the levels of anti-inflammatory cy-
tokines and decreased inhibition of Th17 cells, resulting in liver fibrosis and hepatocellular
carcinoma [51]. Our results suggest that exposure to AFB1 may lead to an increase in BF311
spp. and a decrease in Alistipes spp. populations, and the changes in these bacteria are
related to liver dysfunction in sheep.

4. Conclusions
This study confirmed that oxidative stress, inflammatory injury, apoptosis, and gut
microbiota are involved in the liver injury and liver dysfunction caused by AFB1 exposure
in sheep. The study also offers a valuable reference for future research into the mechanism
underlying the hepatotoxic effects of aflatoxin on sheep.

5. Materials and Methods

5.1. Toxin
AFB1 (>98% pure) was purchased from Pribolab Chemical Inc., Co. (Qingdao, China)
and dissolved in 4% (v/v) ethanol.

5.2. Animals, Exposure Experiment

The study was mainly conducted at Henan Agricultural University’s Xuchang practical
teaching base, China. Twelve Dorper RAMS with an average body weight of 22.34 ± 5.07 kg
were individually identified by ear tag and randomly divided into two groups of three
replicates and two sheep each. The sheep were immunized according to routine procedures.
The control group received 4% ethanol (20 mL) through gavage, while the AFB1 group
received AFB1 (1 mg/kg, dissolved in 20 mL 4% ethanol) (half of LD50) orally [8]. Following
the gavage of AFB1 , there was a 24 h fast from food and water, and surgical sampling was
carried out 24 h later. The animal care and experimental procedures were approved by
the Institutional Animal Welfare and Research Ethics Committee of Henan Agricultural
Toxins 2022, 14, 840 11 of 14

University’s College of Veterinary Medicine (Zhengzhou, China) (Permit No: 17-0126, Year
of approval: 2017). The experimental animals were kept under anesthesia during surgery
and every effort was made to minimize their pain, suffering, and death.

5.3. The Color of Conjunctiva

A handheld colorimeter (#SR-62; Shenzhen 3nh Technology Co., Ltd., Shenzhen,
China) was used to assess conjunctival color prior to before intragastric administration and
surgical sampling. Reflectance spectrometry was used for the color determination using
the CIELab method.

5.4. Sample Collection

Temperature, respiration, and heart rate were measured before and 24 h after intra-
gastric administration. Rectal temperature and heart rate were measured using a mercury-
in-glass thermometer (range 35–42 ◦ C; accuracy ± 0.3) and stethoscope, respectively. By
counting the movement of the abdominal muscles on both sides while breathing, the
sheep’s breathing rate per minute was calculated. Blood samples were drawn from the
jugular vein; serum was isolated through centrifugation at 4 ◦ C for 15 min at 3000× g and
stored at −20 ◦ C. Fecal samples were collected from the sheep rectum before surgery and
kept at −80 ◦ C. The anterior abdominal midline of umbilicus was selected as the surgical
incision. The liver was exposed by laparotomy, about 2 cm × 3 cm × 3 cm hepatic lobules
were collected for liver evaluation, and clamp hemostasis or electrocautery hemostasis
was used. The wound was strictly aseptic debridement; each layer of abdominal wall was
closed and wrapped with elastic bandage. Part of the liver tissue was cut into 1 cm3 pieces
and transferred in liquid nitrogen to the laboratory, where it was preserved at −80 ◦ C for
further analysis. The other portion was fixed for 24 h with 4% paraformaldehyde.

5.5. Serum Biochemistry

To determine the 16 biochemical indexes of the serum, a full-automatic biochemical
analyzer (#SMT-120 V, Seamaty technology Co., Ltd., Chengdu, China) was used, and the
reagent plate was obtained from Chengdu Polytech biological technology Co., Ltd., China.
The following parameters were measured within 2 h: ALB, TP, CK, GLU, AMY, A/G, Ca,
TBIL, AST, ALT, CRE, TG, P, BUN, and GLO.

5.6. Liver Histopathology

Paraformaldehyde-fixed liver tissue samples were washed and dehydrated in ethanol
before being extracted with toluene and embedded in paraffin. For qualitative histological
analysis, tissues were sectioned (5 µm) and stained with hematoxylin and eosin (H&E).
Motic BA600-4 microscope was used to photograph the tissues (Motic, Xiamen, China).

5.7. Detection of Apoptosis

Paraffin slices of liver were dewaxed using xylene and anhydrous ethanol, then
incubated in a 37 ◦ C incubator for 22 min with protease K. TDT enzyme and dUTP were
used to incubate in incubator at 37 ◦ C for 2 h before dropping DAPI dye and incubating for
10 min at room temperature. A fluorescent microscope was used for photo imaging (Nikon
Eclipse C1, Nikon, Japan). Apoptosis was indicated by green fluorescence.

5.8. Liver Function of Tissue

The liver tissue was homogenized to 10% by the dilution solution, and the activity of
general marker enzymes like ALT, AST, and alkaline phosphatase (AKP) enzymes in liver
tissue were assessed using kits (Nanjing Jiancheng Bioengineering Factory, Nanjing, China).

5.9. Liver Antioxidant Abilities

The activities of superoxide dismutase (SOD) and catalase (CAT), and the concen-
tration of total antioxidant capacity (T-AOC) and malondialdehyde (MDA) in the liver
Toxins 2022, 14, 840 12 of 14

tissue supernatant were detected by the kit (Nanjing Jiancheng Bioengineering Institute,
Nanjing, China).

5.10. Extraction of Total RNA and Quantitative Real-Time PCR Analysis

The CFX96 real-time PCR detection system was used for real-time quantitative PCR
(Bio-Rad, Munich, Germany). TRIZOL reagent (Takara Biotechnology Co., Ltd., Dalian,
China) was used to extract total RNA from liver tissue, and Superscript II reverse tran-
scriptase was used to prepare first strand cDNA (Roche, Basel, Switzerland). SYBR Green I
PCR Master Mix (Vazyme Biotech Co.,Ltd, Nanjing, China) was used to determine mRNA
levels, which were then calculated using the 2−∆∆CT method. Sangon Biotech (Shanghai,
China) designed specific primers (Table S1) for inflammatory genes (TNF-α, IL-1β, IL-6, and
IL-10), antioxidant genes (SOD-1, SOD-2), and apoptosis genes (Bcl-2, Bax, and Caspase-3)
based on the NCBI database sequence (Table S1).

5.11. Extraction of Faecal DNA, PCR Amplification, and Illumina Sequencing

Following the manufacturer’s instructions, total fecal DNA was extracted using the
Fast DNA SPIN extraction kits (MP Biomedicals, Santa Ana, CA, USA). To amplify the
V3-V4 region of the 16S rRNA gene, forward primer 338F and reverse primer 806R were
used. QIIME2 dada2 and R package (v3.2.0) were primarily used for sequence data analysis.
The National Center for Biotechnology Information Sequence Read Archive now contains
the sequence information for the 16S rRNA gene obtained in our study (PRJNA844551).

5.12. Statistical Analysis

To ensure accuracy and reproducibility, every experiment was performed at least
three times. Graphpad Prism (version 7.0) was used to analyze all of the data, which was
expressed as means ± standard deviation (SD). The significance of any differences between
the experimental groups was assessed using Student’s t-test. Statistics were considered
significant at p < 0.05.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/toxins14120840/s1, Table S1: Primer sequences for qPCR analysis;
Table S2: Effects of AFB1 exposure on body temperature, breathing, and heart rate of sheep; Table S3:
Effects of AFB1 exposure on serum biochemistry of sheep. Figure S1: Effects of AFB1 exposure on
conjunctival color.
Author Contributions: Conceptualization, Y.L., H.W., X.B., H.D. (Hongyu Dai), X.B., F.L. and H.D.
(Haiju Dong); methodology, Y.S., Y.L., S.Z., H.W., X.B., G.C., H.D. (Hongyu Dai), F.L. and H.D. (Haiju
Dong); software, Y.S.; validation, S.Z.; data curation, Y.S. and Y.L.; writing—original draft preparation,
Y.S.; writing—review and editing, S.H. and H.D. (Haiju Dong).; supervision, H.D. (Hongyu Dai), F.L.
and H.D. (Haiju Dong); funding acquisition, H.D. (Haiju Dong). All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the Henan Province Project for Tackling Key Problems in
Science and Technology (92102110079 and 222102110053), the Young Backbone Teachers Project
of Colleges and Universities in Henan Province (2018GGJS031), and the National Natural Science
Foundation of China (31402187).
Institutional Review Board Statement: All animals used in this experiment were cared for in accor-
dance with the Institutional Animal Welfare and Research Ethics Committee of Henan Agricultural
University’s College of Veterinary Medicine (Zhengzhou, China) (Permit No: 17-0126, date of ap-
proval: 26 January 2017).
Informed Consent Statement: Not applicable.
Data Availability Statement: The National Center for Biotechnology Information Sequence Read
Archive now contains the sequence information for the 16S rRNA gene obtained in our study
(PRJNA844551).
Conflicts of Interest: The authors declare no conflict of interest.
Toxins 2022, 14, 840 13 of 14

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