DISCUSSION
To evaluate the CEGA model as an in vivo NAM, it is critical to
assess target tissue exposure. Since the injections in CEGA are done in
the air space of fertilized eggs, there is a layer of eggshell membrane
which xenobiotics must cross in order to reach the target tissue, which
is liver. To demonstrate target tissue exposure and sufficient
metabolism after reaching target tissue multiphoton microscopy,
metabolite analysis, and genomic studies were conducted.
For multiphoton microscopy, acridine orange was selected as an
appropriate fluorescence dye since the molecular weight of acridine
orange is in the range of B(a)P. The results indicated that even though
the chemical was injected into the airspace, it penetrated the eggshell
membrane and reached the liver (Figure 2), resulting in sufficient liver
uptake.
To study metabolism in CEGA, B(a)P was used as a chemical of choice,
since it is known to produce its genotoxic effect post metabolic
activation. Moreover, previous studies in CEGA confirmed that B(a)P
forms DNA adducts and DNA strand breaks in the livers of chicken
embryo-fetuses (Williams et al., 2014). B(a)P-7,8
dihydrodiol-9,10-epoxide is the reactive metabolite known to covalently
bind to DNA forming adducts and producing adverse effects (Figure 1).
Additionally, metabolism of B(a)P has been well documented in animal
studies, which allows for comparison between the metabolites formed in
the livers of chicken embryo-fetuses and other species. The
characterization of metabolites formed in CEGA following exposure to
B(a)P confirmed that the majority of these six metabolites were also
observed in the rat (Figure 4), Two additional metabolites were formed
in the chicken livers. These were identified as M378 and M300, and
formed at 7.2 and 2.1%, respectively. M378 metabolite had an additional
of three oxygen molecules (3O), removal of two hydrogen groups (-2H) and
addition of one sulfate group (SO3), whereas M300 metabolite had
additional three oxygen (3O) on B(a)P ring. However, M378 formation is
also justified and follows similar pathway as mentioned in IARC document
(IARC 1983) which states that B(a)P is metabolized initially by the
microsomal CYP systems to several arene oxides. Once formed, these arene
oxides may rearrange spontaneously to phenols, undergo hydration to the
corresponding trans-dihydrodiols in a reaction catalyzed by microsomal
epoxide hydrolase, or react covalently with GSH, either spontaneously or
in a reaction catalyzed by cytosolic GST (IARC 1983).
Overall, the findings in the metabolism study confirmed that B(a)P
metabolism in CEGA aligns with the established metabolic pathway in
rodents (Decker et al. 2009). Another similarity was observed with the
animal study of orally administered B(a)P in F344 rats, which found a
half-life of B(a)P in rat liver to be 12 hours, suggesting that
unmodified/unmetabolized B(a)P will be 100% converted to its
metabolites 24 hours post-exposure (Ramesh et al., 2001). In CEGA, 100%
of administered B(a)P was converted to its relevant metabolites. The
toxic precursor, (B(a)P-7,8-dihydridiol), was formed at 23.1% 48-hour
post treatment in embryo-fetal chicken livers. In rat liver, however,
B(a)P-7,8-dihydridiol was only present at 10% 48-hour post
administration with its peak liver concentration at ~30%
24-hour post treatment after single dose of 100 mg/kg bw of B(a)P
(Ramesh et al., 2001). Formation of the reactive metabolite, B(a)P-7,8
dihydrodiol-9,10-epoxide, which in CEGA was formed at 20%, is likely to
be responsible for formation of DNA adducts observed in the embryo-fetal
chicken livers (Williams et al., 2014). The difference in the amount of
metabolite formed in CEGA and rodent study mentioned above may also be
due to single oral administration in Ramesh et al., 2001 study as
compared to three different dose in CEGA studies.
The analyses of the differential gene expression in the embryo-fetal
chicken livers following exposure to B(a)P also confirmed that the
compound upregulated the expression of genes responsible for its
bioactivation (Tables 2 and 3, Figure 7). Specifically, out of 10
significantly deregulated genes with >log 2-fold changes,
three were involved in the activity of CYP1A1 and CYP1A2 isoenzymes.
Other identified genes were also involved in the activity of DNA strands
break mediated gene H2AX and DNA repair mediated gene RNA polymerase II.
These results support the conclusion that B(a)P at a total dose of 250
ug/ egg upregulates expressions of CYP1A genes which affects the
activity of CYP1A1 (Suppl. Figure 1), leading to formation of reactive
metabolite, BPDE, resulting in DNA damage, and activating DNA repair
mechanisms.
In addition to the upregulation of genes involved in CYP1A1 activity, an
increase was also observed in the expression of genes which regulate
CYP1A1 by negative feedback loop. Specifically, upregulation of AhR
repressor and TCDD Inducible Poly (ADP-Ribose) Polymerase (TiPARP) genes
(Table 2) can negatively regulate CYP1A1 activity. This also add to the
fact that with B(a)P treatment there was significant increase in CYP1A1
activity, which is a critical enzyme for metabolizing B(a)P leading to
toxic metabolite, but gene regulating CYP1A1 expression by negative
feedback mechanism were also present in chicken livers.
Similarities between the expressions of CYP1A1 and CYPA2 genes in the
embryofetal chicken livers following dosing with B(a)P were observed
with the published data in rodents and human cells. For example,
expressions of CYP1A1 and CYP1A2 genes in the livers of Wistar rats that
received B(a)P at a single dose of 150 mg/kg bw by oral gavage was
significantly increased by 2990 and 27.7 folds, respectively(Dračínská et al., 2021) . In the study with human hepatocellular
carcinoma cell line (HepG2) which was incubated with 2 µM of B(a)P,
CYP1A1 showed a 93-fold and 79-fold increase in expression on microarray
12- and 24-hours post-dosing, respectively, whereas RNA-seq demonstrated
a 199-fold (at 12 hours) and 214-fold (at 24 hours) increases in CYP1A1
expression (Van Delft et al.; 2012). In the human tissue organoid
cultures, differentiated liver had significantly higher (24-fold) CYP1A1
levels compared to undifferentiated samples, at basal level. After
exposure to 50 µM of B(a)P, induction of CYP1A1 in differentiated liver
organoids was around double compared to that in undifferentiated
organoids (~4500- and 2000-fold, respectively), relative
to control. At 12.5 µM, a 287-fold change compared to undifferentiated
control was observed only in differentiated organoids. Induction of
CYP1A1 was also significant at both concentrations of B(a)P compared to
differentiated control (Caipa Garcia et al., 2023). These results are
consistent with findings in CEGA which demonstrated a fold change of
1024 folds for CYP1A1 activity combined (ENSGALG00000013402 and
ENSGALG00000001325) and 16 folds for CYP1A2 activity (Table 2).