INTRODUCTION
Carcinogenic chemicals can be broadly classified based on their mode of
action, with a major focus on genotoxic and non-genotoxic mechanisms.
Genotoxic carcinogens directly interact with the genetic material of
cells, causing mutations, chromosomal fragmentation, or rearrangements.
These alterations can disrupt normal cellular functions, leading to
uncontrolled cell proliferation and ultimately cancer.
Genotoxicity of chemicals can be evaluated in various in vitro and in vivo assays, which are mainly designed to evaluate
mutagenicity potential, chromosomal damage, and DNA damage /repair
pathways interruption. Due to the recent restrictions in the use ofin vivo genotoxicity assays, there is a need for biologically
relevant new approach methodologies (NAMs) to be used as animal
alternatives for evaluating genotoxic potential of chemicals that hadin vitro positive results.
The Chicken and related Turkey Egg Genotoxicity Assays (CEGA and TEGA,
respectively) (Williams et al., 2014; Iatropoulos et al., 2017; Kobets
et al., 2018b; Kobets et al., 2016; 2018a), were developed as
metabolically competent (Kobets et al., 2018b; Perrone et al., 2004)
NAMs for genotoxicity screening to potentially replace short-termin vivo studies required for human safety assessment. CEGA uses
fertilized, specific pathogen free eggs from the white leghorn chicken
of undetermined sex. Since the termination of the embryos in CEGA is
conducted on incubation day 11, at least 10 days before hatching,
discomfort to the organism is precluded, as the nervous system of the
embryos is not completely developed (Hughes 1953). Thus, in compliance
with Animals (Scientific Procedures) Act 1986, CEGA is not considered to
be an animal model.
CEGA evaluates two different endpoints, DNA adducts by the means of the
NPL assay (Phillips and Arlt, 2014; Randerath et al., 1981; Reddy and
Randerath, 1986) and DNA strand breaks using the alkaline single cell
gel electrophoresis (comet) assay (Brendler-Schwaab et al., 2005; OECD,
2016; Tice et al., 2000). These are indicative of DNA damage produced by
either direct or indirect mechanisms. Both techniques are widely used
for the evaluation of chemical-induced DNA damage (Himmelstein et al.,
2009 and also makes it possible to elucidate the mode of action of
chemical carcinogens. Additionally, fetal avian livers express majority
of the phase-I and phase-II biotransformation enzymes which can detect
chemicals inducing DNA damage post metabolic transformation (Kobets et
al., 2018b; Perrone et al., 2004; Rifkind et al., 1979) and they can
also efficiently mimic detoxification of chemicals similar to rodent
models.
Genotoxicity can be induced by direct DNA activity of the parent
chemical and/or its metabolite. As such, metabolism plays a crucial role
in the bioactivation of many chemicals. This process is often required
for the formation of reactive electrophilic intermediates that can then
directly react with DNA (Kobets et al., 2019). Bioactivation of
different classes of chemicals may differ and produced metabolites may
interact with different sites on macromolecules including DNA.
Since many in vitro systems lack an intrinsic ability to
metabolize chemicals, the induced rat liver S9 fraction is used as an
exogenous metabolic activation system (Ames et al, 1973; Paolini et al,
1997). However, this exogenous source of metabolic enzymes does not
include those that are important for phase II detoxification. Hence,
current in vitro testing systems generate high number of
misleading outcomes in testing and prediction of carcinogens (Kirkland
et al., 2007).
For analysis of the chicken egg liver response to a xenobiotic, a
polycyclic aromatic hydrocarbon, benzo[a]pyrene (B(a)P) was chosen.
Many of the chemicals that belong to this group are genotoxic
carcinogens (Urwin et al., 2024). Carcinogenic activity of B(a)P
involves activation of the Aryl hydrocarbon receptor (AhR), which in
turn binds to AhR nuclear translocator and induces the expression of
genes involved in B(a)P bioactivation and detoxification. These genes
are the cytochrome P450 (CYP) genes CYP1A1, CYP1A2, CYP1B1, as well as
glutathione transferase (GST) and Uridine diphosphate
(UDP)-glucuronosyltransferase (UGT-1). In order to exhibit genotoxicity,
B(a)P requires oxidation by phase I CYP1A1 into B(a)P-7,8-epoxide, which
through hydration by microsomal epoxide hydrolase is metabolized to
B(a)P-7,8-dihydrodiol (BPD) (Figure 1). BPD is then metabolized to
B(a)P-7,8-dihydrodiol-9,10-epoxide (BPDE) by second CYP reaction (Kim et
al., 2005). BPDE contains an epoxide ring which is highly reactive with
DNA in a time dependent manner. In vitro , B(a)P consistently
produced negative outcomes in mutagenicity and clastogenicity studies in
the absence of metabolic activation, only demonstrating positive
outcomes in the presence of exogenous S9 fraction (EPA, 2017).