Evaluation of the effect of tartrazine on the offspring rats in an in vivo experimental model

Abstract

Tartrazine, an azo dye prevalent in pharmaceuticals and food items, was investigated for its impact on fetal development, specifically examining visceral and skeletal abnormalities in rat offspring exposed to daily oral doses throughout pregnancy. Fourteen pregnant rats were randomly assigned to control and tartrazine groups (seven animals each), with tartrazine administered via oral gavage at 7.5 mg/kg throughout gestation. Offspring were categorized by gender for histopathological and genetic analysis of visceral structures. Bone quality and fracture resistance assessments involved micro‐CT, Raman spectroscopy, and biomechanical testing. Results highlighted distinct internal organ tissue differences in the tartrazine group, notably increased hemorrhagic and inflammatory cell infiltration, degeneration, and vacuolization compared to controls. Gender‐specific alterations in mRNA levels of IL‐6, IL‐1β, TNF‐α, and TRPM2 genes (p < .001) were also noted. Moreover, tartrazine‐exposed groups exhibited reduced trabecular thickness, bone volume, and significant alterations in bone matrix composition and quality alongside significant decreases in fracture resistance (p < 0.05). This study concludes that intrauterine exposure to tartrazine can result in adverse impacts on organ and bone development in rat offspring.

Full text

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Food Sci Nutr. 2024;12:9162–9174.wileyonlinelibrary.com/journal/fsn3
Received: 28 May 2024
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Revised: 30 August 2024
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Accepted: 10 September 2024
DOI: 10.1002/fsn3.44 85
ORIGINAL ARTICLE
Evaluation of the effect of tartrazine on the offspring rats in an
in vivo experimental model
Osman Öztürk1 | Yusuf Dikici2,3 | Öznur Gür4 | Mert Ocak5 |
ZüleyhaDoğanyiğit6 |AslıOkan6 |EvrimSunaArıkanSöylemez7 |
ŞükrüAteş8 | Sümeyye Uçar9 | Mustafa Unal4,10,11 |SeherYılmaz2,8
1Department of Pediatrics, Faculty of Medicine, Yozgat Bozok University, Yozgat, Turkey
2Depar tment of Mechanical and Aerospace Engineering, Case Western Reserve University, Cleveland, Ohio, USA
3Depar tment of Mechanical Engineering, Faculty of Engineering, Architec ture and Design, Bartın University, Bartın, Turkey
4Depar tment of Mechanical Engineering, Karamanoglu Mehmetbey University, Karaman, Turkey
5Depar tment of Basic Medical Sciences, Anatomy, Faculty of Dentistry, Ankara University, Ankara, Turkey
6Depar tment of Histology and Embriology, Faculty of Medicine, Yozgat Bozok University, Yozgat, Turkey
7Depar tment of Medical Biology, Faculty of Medicine, Afyonkar ahisar Health Sciences University, Afyonkarahisar, Turkey
8Depar tment of Anatomy, Faculty of Medicine, Yozgat Bozok Universit y, Yozgat, Turkey
9Depar tment of Anatomy, Faculty of Medicine, Erciyes University, Kayseri, Turkey
10Department of Bioengineering, Karamanoglu Mehmetbey University, Karaman, Turkey
11Depar tment of Biophysics, Faculty of Medicine, Karamanoglu Mehmetbey Universit y, Karaman, Turkey
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2024 The Author(s). Food Science & Nutrition published by Wiley Periodicals LLC.
Correspondence
Seher Yilmaz, Department of Mechanical
and Aerospace Engineering, Case Western
Reserve Univer sity, Cleveland 4 4106, OH,
USA. Department of Anatomy, Facult y of
Medicine, Yozgat Bozok Univesity, Yozgat,
Tur ke y.
Email: seher.yilmaz@yobu.edu.tr and
sxy941@case.edu
Abstract
Tartrazine, an azo dye prevalent in pharmaceuticals and food items, was investigated
for its impact on fetal development, specifically examining visceral and skeletal
abnormalities in rat offspring exposed to daily oral doses throughout pregnancy.
Fourteen pregnant rats were randomly assigned to control and tartrazine groups
(seven animals each), with tartrazine administered via oral gavage at 7.5 mg/kg
throughout gestation. Offspring were categorized by gender for histopathological
and genetic analysis of visceral structures. Bone quality and fracture resistance
assessments involved micro- CT, Raman spectroscopy, and biomechanical testing.
Results highlighted distinct internal organ tissue differences in the tartrazine group,
notably increased hemorrhagic and inflammatory cell infiltration, degeneration, and
vacuolization compared to controls. Gender- specific alterations in mRNA levels of I L-
6, I L- 1β, TNF- α, and TRPM2 genes (p < .001) were also noted. Moreover, tartrazine-
exposed groups exhibited reduced trabecular thickness, bone volume, and significant
alterations in bone matrix composition and quality alongside significant decreases
in fracture resistance (p < 0.05). This study concludes that intrauterine exposure
to tartrazine can result in adverse impacts on organ and bone development in rat
offspring.

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1 | INTRODUC TION
Tartrazine, a synthetic yellow food dye widely used in industries like
food and textiles (Ismail & Rashed, 2022), se r ves to enh anc e and pre-
serve flavor, color, and texture in food items (Amchova et al., 2015).
Such artificial food colors undergo approval from the Food and Drug
Administration (FDA) before integration into the food industry (Dey
& Nagababu, 2022), with tar trazine ranking among the most prev-
alent dyes (El- Desoky et al., 2022). To regulate concentrations in
food, cosmetics, and pharmaceuticals, the acceptable daily intake
(ADI) for humans ranges from 0 to 7.5 mg/kg body weight (Pressman
et al., 2017).
Tartrazine, a common additive, is found in various daily food
products such as chips, fruit juices, cakes, cornflakes, soups, can-
dies, ice creams, and gums. Additionally, it is utilized in shampoos
and hair care items (Barciela et al., 2023; Pay et al., 2023). Studies
have associated tartrazine with toxic effects stemming from met-
abolic processes that transform its azo bond (Al- Seeni et al., 2018;
Haugabrooks & Hayes, 2023). Some research also hints at poten-
tial carcinogenic effects, particularly concerning intestinal ab-
sorption and these effects appear to vary based on an individual's
age, gender, and genetic disposition (Hanna et al., 2023; Sambu
et al., 2022).
The metabolites of tartrazine instigate the production of re-
active oxygen species (ROS), precipitating oxidative stress that
exerts biochemical and cellular repercussions on organs such
as the liver, stomach, and kidneys (Albasher et al., 2020; Amin
et al., 2010; El Golli, 2016; Visweswaran & Krishnamoorthy, 2012).
These metabolites, when transformed into aromatic amines, can
traverse both the blood–brain and placental barriers. Research
indicates potential teratogenic effects from food additives con-
sumed during pregnancy (Hashem et al., 2019). There are also
several studies on embryotoxic substances during pregnancy, ex-
ploring fetal bone development using diverse staining methods
(Tokpinar et al., 2024; Yılmaz et al., 2020). Previously, embryo-
toxic and teratogenic effects of tartrazine have been investigated
in various animal models. For example, previous studies have
shown that tartrazine exhibits teratogenic effects on rats and ze-
brafish embryos (Gupta et al., 2019; Hashem et al., 2019; Sambu
et al., 2022). Additionally, it has been reported to have genotoxic
potential and induce DNA damage in the liver, kidneys, and leu-
kocytes of rats (Balta et al., 2019; Khayyat et al., 2017; Nasri &
Pohjanvirta, 2021). Additionally, Khayyat et al., 2017 found that
tartrazine may cause structural and functional aberrations, as
well as severe histopathological and cellular alterations in the
liver and kidney tissues of rats. These findings suggest potential
risks associated with tartrazine exposure during embryonic devel-
opment and its adverse effects on the physiological functions of
various organs. Furthermore, tartrazine has been linked to oxida-
tive stress, elevated hepatocellular enzyme activities, and alter-
ations in kidney biomarkers, indicating potential toxicity in adult
rats (El Golli, 2016). Moreover, the impact of tartrazine on learn-
ing and memory functions in animals has been studied, suggest-
ing potential neurotoxic effects (Gao et al., 2011). Moreover, Pan
et al., 2011 characterized the interaction between tartrazine and
serum albumins, revealing conformational and microenvironmen-
tal changes that could potentially impact the physiological func-
tions of these proteins.
In the context of bone strength and quality in offspring born to
pregnant rats exposed to tartrazine, there is currently no direct ev-
idence linking tartrazine to bone tissue effects. The emerging un-
derstanding of its teratogenic and toxic effects on various organs
raises concerns about its potential impact on both bone develop-
ment and several organs. Hence, there exists a discernible neces-
sity for inquiries aimed at unraveling the conceivable impacts of
tartrazine on bone fracture resilience and quality, considering the
complex interplay between embryonic development and bone syn-
thesis. Consequently, our objective was to scrutinize the influence
of tartrazine on bone integrity alongside its effects on other cor-
related organs in the progeny of pregnant rats subjected to tartra-
zine exposure.
2 | MATERIALSANDMETHODS
2.1 | Ratsandexperimentaldesign
The study utilized 2- month- old female Sprague Dawley rats (N = 14)
weighing 250–300 g obtained from Erciyes University Experimental
Animal Center. The appropriateness of this investigation involving
animal experimentation adhered to the guidelines outlined
in decision 24/008 issued by the Erciyes University Animal
Experiments Local Ethics Committee. The rats were fed a standard
rat diet. Pregnant rats were accommodated in meticulously arranged
chambers equipped with automated heating systems, maintaining a
stable temperature of 21°C, and subjected to alternating 12- hour
periods of light and darkness for the duration of the research.
Control Group (n = 7): Physiological saline was administered by
oral gavage during pregnancy.
Tartrazine Group (n = 7): Tartrazine at a dose of 7.5 mg/kg/day was
administered by oral gavage (El Imam & Abd El Salam, 2018; Ismail
& Rashed, 2022) during pregnancy. After the gestation period, the
offspring born from the rats were separated by gender and placed
in individual. At 1 month of age, they were sacrificed under ketamine
xylazine anesthesia. In our study, six male and six female offspring
with similar measurements were used for each test.
KEYWORDS
azo dye, bone microstructure, bone quality, Raman spectroscopy, tartrazine

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2.2 | Samplecollectionandtissuepreparation
After sacrifice, heart, lung, kidney, and liver tissues were collected
from each animal for histology and Rt- Pcr analysis. Femur bones
were also collected, cleaned with physiological saline (PBS), and
stored at −20°C with PBS to prevent dehydration. Samples from
each group for each parameter were used for Raman spectroscopy,
biomechanical testing, and micro- CT analysis. The experimental
setup and investigated parameters are shown in Figure 1.
2.3 | Histologicalanalysis
After removing heart, lung, kidney, and liver tissues from both fe-
male and male animals, they were placed in a 10% formaldehyde
solution and fixed for 8 to 12 hours (Doğanyiğit et al., 2020). After
fixation, the tissues were submerged in flowing water for the dura-
tion of the night. The routine histological paraffin embedding pro-
cess was performed on a day. Five- micrometer- thick sections were
taken from each paraffin block sample. The sections were passed
through a xylene and decreasing alcohol series and stained with he-
matoxylin and eosin. The study evaluated glomerular degeneration,
vacuolization in tubulointerstitial injuries, hemorrhage, and infiltra-
tion of inflammatory cells in each animal (Inandiklioglu et al., 2021).
In liver samples, sinusoidal expansion, bleeding, and apoptotic
hepatocytes were evaluated (Doğanyiğit et al., 2020), while in heart
samples, bleeding and irregular myocardial fibers were evaluated
(Akyuz et al., 2023). In lung samples, hemorrhage and mononuclear
cell infiltration were evaluated. Damage rates for each category
were scored as follows: 3 severe, 2 medium, and 1 light. No changes
were obse r ved in som e cas es. Th e tis sue s were analyz ed histopatho-
logically under the Olympus BX53 light microscope.
2.4 | Geneticanalysis
Total RNA was extracted from the hearts, lungs, livers, and kidneys
of the tartrazine- exposed rats using PureZole reagent (Biorad, USA)
following the manufacturer's protocol. The quantity and quality
of RNA in each sample were assessed employing a Nanodrop ND-
1000 spectrophotometer V3.7. The RNA specimens were preserved
at −80°C until required. Subsequently, all RNA samples underwent
reverse transcription into cDNA utilizing 1 μg of total RNA (iScript
Reverse Transcription Supermix, Biorad, USA). Real- time PCR was
used to analyze the expression of IL- 6, IL- 1β, TNF- α, and TRPM2 mR N A.
The Step- One- Plus Thermocycler (Applied Biosystems) was used for
amplification in a total reaction volume of 20 μL, with cDNA, site-
specific primers (Oligomer Biotechnology, Ankara), SsoAdvanced
Universal Inhibitor- Tolerant SYBR Green Supermix (Biorad, USA),
and nuclease- free water. GAPDH was used as an internal control.
FIGURE 1 Schematic representation of the experimental design and the parameters examined (Biore nder. com).

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The primer sequences for TNF- α, IL- 1β, and IL- 6 were designed as
previously mentioned (Iwashita et al., 2014), while those for TRPM2
were designed as previously mentioned (Cook et al., 2010).
Rat- IL- 6 F: 5′- TCCTACCCCAACT TCCAATGCTC- 3′
Rat- IL- 6 R: 5′- TTGGATGGTCTTGGTCCTTAGCC- 3′
Rat- IL- 1 β F: 5′- CACCTCTCA AGCAGAGCACAG- 3′
Rat- IL- 1 β R: 5′- GGGTTCCATGGTGAAGTCA AC- 3′
Ra t- TNF- α F: 5′- AAATGGGCTCCCTCTCATCAGTTC- 3′
Ra t- TNF- α R: 5′- TCTGCTTGGTGGTTTGCTACGAC- 3′
Ra t- TRPM 2 F:5′GAAGGAAAGAGGGGGTGTG- 3′
Ra t- TRPM 2 F: 5′ CATTGGTGATGGCGTTGTAG- 3′
Rat- GAPDH F: 5′- GAGGACC AGGTTGTCTCCTG- 3′
Rat- GAPDH R: 5′- GGATGGAATTGTGAGGGAGA- 3′
We used the following RT- PCR protocol: for IL- 6 , I L- 1β, TNF- α:
98°C for 3 min of initial denaturation followed by 40 cycles of 98°C
for 15 s and 61°C for 30 s, and for TRPM2: 98°C for 30 s initial de-
naturation followed by 40 cycles of 98°C for 5 s and 58°C for 30 s.
Melting curve analysis was performed for confirmation of single-
product amplification at the end of the PCR. 65°C–95°C, 0.5°C in-
crements at 5 s/step. Each run has been performed triplicate.
2.5 | Ramanspectroscopy
After the specimens had fully thawed at room temperature, each
specimen's long axis was carefully aligned parallel to the primary
laser polarization axis. Subsequently, we acquired three Raman
spectra per specimen, ranging between 750 and 1800 cm−1. These
spectra were obtained as an average of ten consecutive spectra
per spot, with a 5- second acquisition duration, utilizing a 20×
objective (NA = 0.40) via a 785 nm Raman micro- spectroscopy (Invia,
Renishaw, UK).
Following the established protocols (Unal et al., 2016, 2018,
2021), we calculated various bone composition parameters from
the Raman spectroscopy data based on peak intensities (Figure 4a).
These parameters include the mineral- to- matrix ratio (ν1PO4/Amide
I), type- B carbonate substitution (CO3/ν1PO4), crystallinity (the re-
ciprocal of the line- width of the ν1PO4 peak at half- maximum; 1/
FWHM), collagen cross- links/matrix maturity ratio (~I1670/I1690),
hydroxyproline to proline ratio (Hyp/Pro), and the ~I1670/I164 0 ratio,
indicative of collagen conformational changes (Figure 4a) (Unal
et al., 2016).
2.6 | Micro-CT
Femur samples taken from female and male offspring were fixed
with a thin paraffin block. It was aligned in the same direction in the
plastic tube for micro- CT scanning. An isotropic voxel size of 20 μm
in a 35 mm field of view was achieved by adjusting the resolution
parameters.
Adjusted images were taken on each sample (Bruker Skyscan
1275, Kontich, Belgium). Then, images were obtained through bone
tissue with an isotropic voxel size of 10 μm using 40kv, 250 μA, ex-
posure time 49 ms, rotation step 0.2, and 360O rotation in a high
resolution 23 mm field of view. Axial, coronal, and sagittal images of
each sample were examined using Dataviewer software (Skyscan,
Kontich, Belgium). CTAn (Skyscan, Aartselaar, Belgium) software
was used for three- dimensional (3D) volumetric visualization, and
area/volume measurement analysis was performed for micro- CT.
2.7 | Biomechanicaltesting
The versatile testing device TA.XT Plus (Stable Micro Systems, UK)
was used to conduct the three- point bending test. The femurs were
first placed at two spans with a constant span length of 7.5 mm
(Figure 7a) and were loaded to fracture at a rate of 3 mm/min (Creecy
et al., 2020). Force measurements were captured using a 500 N load
cell, and force and displacement values were automatically recorded
using the device's software. The structural- dependent mechanical
properties, such as stiffness, yield, and peak load, were calculated
from the force–displacement curve (Figure 7a). The associated
beam equations (Jepsen et al., 2015) were utilized in estimating the
material properties, including elastic/bending modulus, yield stress,
and maximum stress.
2.8 | Statisticalanalysis
The numerical values of the histopathological findings were analyzed
using a two- way analysis of variance and Sidak's multiple comparison
tests. First, normality of the parameters was tested using a Shapiro–
Wilk test. If the normality assumption was met, a one- way repeated
ANOVA test was applied (Doğanyiğit et al., 2024). The genetic
analysis was performed using REST 2009 V2.0.13 Software (Pfaffl
et al., 2002). Statistical analysis was performed using GraphPad
Prism 9 on results obtained from micro- CT, biomechanical testing,
and Raman spectroscopy. Normality and lognormality tests were
conducted using the Kolmogorov–Smirnov test. If the normality
assumption was met, an unpaired t- test was applied. If the normality
assumption was not met, a Mann–Whitney test was applied. The
statistical significance level for all comparisons was set at p < .05
(Selvan et al., 2024).
3 | RESULTS
3.1 | Histologyresults
Our results revealed increased hemorrhaging in the kidneys
and lungs of male groups exposed to tartrazine. Additionally, in
female renal tissues, there were notable instances of glomerular
degeneration and vacuolization. Statistically significant
differences were found in vacuolization, hemorrhage, and
inflammatory cell infiltration. Notably, tartrazine led to increased

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hemorrhage and mononuclear cell infiltration in male lung tissues.
Moreover, female liver tissues exposed to tartrazine showed a
higher count of apoptotic hepatocytes compared to the control
group. While examining female heart tissues, there was a notable
increase in irregular myocardial fibers compared to the control
group (Figure 2).
3.2 | mRNAanalysisofIL- 6 ,  IL- 1 β,
TNF-α, and TRPM2
Alteration of I L- 6 , IL- 1β, TNF- α, and TRPM2 mRNA levels of each
tissue of each gender were determined according to the mRNA
levels of related control tissues. The mRNA levels of the IL- 6 , I L-
1β, TNF- α, and TRPM2 genes were altered in hearts of females
compared to the control (0.41*, 1.23*, 2.73*, 0.03*; fold regulation
value, respectively, *p < .001). The mRNA levels of the I L- 6 , I L-
1β, TNF- α, and TRPM2 genes were altered in hearts of males
compared to the control (0.7*, 0.4*, 1.8, 2.06*; fold regulation
value, respectively, *p < .001). The mRNA levels of the I L- 6 , I L-
1β, TNF- α and TRPM2 genes were altered in lungs of females
compared to the control (0.46*, 4.99*, 2.19*, 0.58*; fold regulation
value, respectively, *p < .001). The mRNA levels of the I L- 6 , IL- 1β,
TNF- α, and TRPM2 genes were altered in lungs of males compared
to the control (2.38*, 0.23*, 2.73*, 1.85*; fold regulation value,
respectively, *p < .001). The mRNA levels of the I L- 6 , IL- 1 β, TNF- α,
and TRPM2 genes were altered in kidneys of females compared
to the control (4.6*, 1.25, 1.68, 0.03*; fold regulation value,
respectively, *p < .001). The mRNA levels of the IL- 6 , I L- 1β, TNF-
α, and TRPM2 genes were altered in kidneys of males compared
to the control (0.21*, 0.99*, 0.088*, 1.05; fold regulation value,
respectively, *p < .001). The mRNA levels of the IL- 6 , I L- 1β, TNF-
α, and TRPM2 genes were altered in livers of females compared
to the control (0.8, 1.81*, 0.83, 1.45; fold regulation value,
respectively, *p < .001). The mRNA levels of the I L- 6 , IL- 1 β, TNF- α,
and TRPM2 genes were altered in livers of males compared to the
control (0.31*, 0.6, 1.1, 0.34*; fold regulation value, respectively,
*p < .001) (Figure 3).
3.3 | Ramanspectroscopy
Our Raman analysis revealed substantial alterations in bone
composition and quality within the tartrazine- exposed groups
compared to the control, regardless of sex. Specifically, the
mineral- to- matrix ratio (ν1PO4/Amide I) exhibited a significant
decrease in the tartrazine group (Figure 4b), indicating reduced
local tissue mineralization. Concurrently, lower crystallinity values
in the tartrazine group suggested diminished mineral crystal
perfection compared to the control group. Moreover, there was
an observed increase in carbonate substitution in the tartrazine
groups across both sexes, with statistical significance reached
specifically within the female group. This alteration highlights
a notable change in bone mineral quality and maturity due to
tartrazine exposure. Regarding collagen- related Raman properties,
the Hyp/Pro ratio, indicative of collagen maturity and structural
alteration, exhibited a significant increase in the tartrazine group.
Similarly, an increase in the ~I1670/I1640 ratio, reflecting alte rati on in
collagen triple helix st ruc ture in te grity, was note d in th e t art razine
group compared to the control group. Additionally, the mature-
to- immature enzymatic cross- links ratio (~I1670/I1690) significantly
decreased in the tartrazine group, suggesting further structural
changes in collagen. Our findings underscore the significant
impact of tartrazine on bone composition and quality, affecting
both mineralization and collagen structure across the studied
groups (Figure 4b).
3.4 | Micro-CT
Figure 5 displays micro- CT images showcasing the trabecular bone
appearance of the total femur and femoral head across all groups.
The control female and male groups exhibited similar cancellous
bone structures characterized by numerous trabecular connections.
In contrast, the tartrazine- exposed groups displayed numerous
trabecular breaks and significantly larger trabecular porosity.
Figure 6 highlights key findings: Trabecular thickness in the
tartrazine- exposed groups was notably lower compared to the con-
trol group in male (p < .001). In females, this value was also lower in
the tar tr azine- applied group compared to the control, but the differ-
ence was not statistically significant (p > .05). Bone volume was re-
duced in tartrazine- exposed male offspring in contrast to the control
(p < .05). Moreover, trabecular separation was observed to be dimin-
ished in tar trazine- exposed mal e of fspring in com parison to the con-
trol (p < .0001). When bone surface density and trabecular number
values were examined, an increase was observed in both males and
females as a result of tartrazine exposure compared to the control
group. This increase was statistically significant in males (p < .05).
3.5 | Biomechanicaltesting
Our investigation into both structural- and material- level mechanical
properties revealed significant reductions in the mechanical fracture
resistance of femurs due to tartrazine exposure. Notably, stiffness
and its material- level counterpart, elastic modulus, displayed
decreases in the tartrazine- exposed group compared to the control,
irrespective of sex (p < .05). Furthermore, both yield load (structural
level) and yield stress (material level) exhibited decreases in the
tartrazine- exposed groups in contr ast to the cont rol groups (p < .05).
Both peak load and maximum stress also showed considerable
reduction in the tartrazine- exposed groups when compared to the
control group (p < .05) (Figure 7b). Collectively, these mechanical
findings suggest a significant decline in the mechanical properties of
femurs in the tartrazine groups, emphasizing its adverse impact on
bone fracture resistance (Figure 7b).

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FIGURE 2 Legend on next page

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4 | DISCUSSION
Recent studies have reported that the intake and dosage of tartra-
zine, widely used in the food industry, similar to Indole- 3- acetic acid
in agriculture, can cause cellular damage in experimental animals,
potentially leading to organ damage (Haridevamuthu et al., 2024;
Nayak et al., 2024). Moreover, according to the study published
by Khayyat et al., tartrazine has been linked to potential hepato-
renal and cardiovascular toxicity, causing structural and functional
abnormalities, genotoxic effects, and alterations in physiological
and biochemical parameters (Haridevamuthu et al., 2024; Khayyat
et al., 2017). Studies have also reported severe histopathological
and cellular alterations in liver and kidney tissues, DNA damage,
and changes in antioxidant enzymes, lipid peroxidation, and lipid
profile in animal models (Adele et al., 2020; El- Hakama et al., 2022;
Khayyat et al., 2017). Furthermore, tartrazine has been associated
with damage to the gastric mucosa, as reported previously (Elwan &
Ibrahim, 2019), an d alterat ions in ca rot id an d aorti c sti f fne ss, ind icat-
ing a potential impact on cardiovascular health (Bruno et al., 2017).
Histological examinations of organs revealed increased necrosis and
inflammation in the liver and kidney tissues of experimental animals
exposed to tartrazine, resulting in organ damage. The specific dam-
ages observed in the studies include glomerular necrosis, portal vein
level edema, and inflammation (Demircigil et al., 2023; El- Desoky
et al., 2022; Hoc et al., 2006). Heightened levels of TNF- α, I L- 1 β, and
IL- 6 were reported in brain tissue of rats exposed to tartrazine in
recent studies (Demirkol et al., 2012; Essawy et al., 2023). Based on
the findings, the application of tartrazine to pregnant rats, even at a
FIGURE 2 Hematoxylin and Eosin- stained images of kidney, lung, liver, and heart tissues from both male and female experimental
groups. The images include control female group (a, f, k, p), tartrazine- exposed female group (b, g, l, q), control male group (c, h, m, r), and
tartrazine- exposed male group (d, i, n, s). The magnification is set at 40× with a scale bar of 20 μm. In the kidney images, the yellow arrow
indicates hemorrhage, the black arrow points to glomerular degeneration, the white arrow highlights vacuolization, and the blue arrow
shows infiltration of inflammatory cells. For lung images, the yellow arrow signifies hemorrhage, while the blue arrow denotes inflammatory
cell infiltration. The liver image showcases black arrows indicating apoptotic hepatocytes, and the heart image highlights irregular myocardial
fibers. Histology score bar charts for kidney (e), lung ( j), liver (o), and heart (t) are provided, expressed as ± SD. The statistical analysis
conducted involves two- way ANOVA and Tukey's multiple comparison tests denoted as follows: (a, α – p < .05 different from control female
group), (b, β, μ, Ω – p < .05 different from tartrazine female group), and (c, γ, χ, ε – p < .05 different from control male group).
FIGURE 3 The results of real- time PCR analysis. Relative mRNA expressions of IL- 6 , IL- 1 β, TNF- α, and TRPM2 genes in heart (a), lung (b),
kidney (c), and liver (d) tissues exposed to tartrazine were given as fold regulation level, log(10). *Represents the significance of p < .001
compared to control. GAPDH is a reference gene for normalization.

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low dose of 4.5 mg/kg, resulted in regression in heart, lung, and bone
development in fetal organs, as well as impaired fetal growth com-
pared to the control group (Hashem et al., 2019). Herein our results
also clearly indicate the adverse effects of tartrazine on the heart,
lungs, kidneys, and liver tissues of offspring born to pregnant rats
exposed to tartrazine (Figures 2 and 3).
Additionally, studies have shown that external intake of sub-
st ance s suc h as drugs an d foo d dye s during pr egn a ncy can affec t em-
bryonic development during the organogenesis period in mammals,
crossing the placental barrier (Atay et al., 2019; Booth et al., 2022).
Various studies have explored the safety concerns and technological
applications of food colorants, including azo dyes (Jiang et al., 2020;
Pay et al., 2023). However, there remains a gap in understanding
their specific effects on bone quality and fracture resistance.
To our current understanding, this investigation represents the
inaugural examination assessing the impacts of tartrazine on the
quality of bone tissue and its resistance to fractures. This current
study reports the adverse effects of tartrazine on offspring born
from pregnant rats exposed to tartrazine, impacting bone tissue, re-
gardless of gender. More specifically, our results indicate that rats
exposed to tartrazine during pregnancy exhibit a decrease in bone
composition and quality compared to the control group. Raman
spectroscopy- based assessment of bone matrix provides compre-
hensive information on bone composition, including both mineral
and collagen (Unal et al., 2021). Our results suggest significant ad-
verse effects of tartrazine on skeletal development, affecting not
only mineral amount per organic matrix but also mineral quality
(Figure 4b). Additionally, collagen quality was also diminished due
to exposure to tartrazine during pregnancy (Figure 4b). The ad-
verse effects of tartrazine on bone tissue are not limited to bone
composition level. Our findings further demonstrate that bone mi-
crostructural properties, which play a crucial role in bone fracture
resistance (Hoc et al., 2006), are impacted by tartrazine exposure
during pregnancy (Figures 5 and 6). Exposure to tartrazine during
gestation can have deleterious effects on the bone composition and
microstructure of rat offspring, resulting in reduced bone fracture
resistance. This assertion is corroborated by the diminished met-
rics of diverse bone mechanical characteristics noted in the group
exposed to tartrazine, in contrast to the control group (Figure 7b).
These findings emphasize the need for further research to evaluate
the potential link between tartrazine and reduced bone fracture re-
sistance and quality. Tartrazine may affect bone quality and fracture
resistance through various mechanisms, including the generation of
ROS and oxidative stress. Several studies have shown that tartra-
zine induces ROS and oxidative stress (Albasher et al., 2020; Amin
et al., 2010; El Golli, 2016; Visweswaran & Krishnamoorthy, 2012).
In fact, ROS has been extensively studied in bone biology research
due to their impact on bone fracture and health. They play a role
FIGURE 4 (a) A typical Raman
spectrum of bone and calculated Raman
spectroscopy- based bone quality
properties. (b) Both mineral and collagen
quality properties are significantly
affected by tartrazine. The statistical
significance level for all comparisons was
set at p < .05.

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ÖZTÜRK et al .
in physiological processes such as osteoclastic activity in bone re-
sorption, as opposed to osteoblastic activity, which is active in bone
remodeling (Filip et al., 2018; Sugumaran et al., 2023). Studies have
demonstrated that ROS are involved in osteoclast differentiation
and bone resorption, as well as in osteoblast function and bone
formation (Agidigbi & Kim, 2019; Schoppa et al., 2022). ROS have
been linked to the regulation of osteoclastogenesis and osteobl as to-
genesis, indicating their involvement in bone remodeling processes
(Domazetovic et al., 2017; Zhu et al., 2022). Additionally, excessive
ROS generation has been shown to cause oxidative stress, which can
FIGURE 5 Micro- CT scan of the femur (a), the micro- CT images show that the trabeculae and trabecular separating head of the femur
and greater trochanter can be seen in transverse section (b).
FIGURE 6 Bone volume (mm3) (a), bone surface density (b), trabecular thickness (mm) (c), trabecular separation (d), trabecular number (e).
Values are given as the means ± SD. The statistical significance level for all comparisons was set at p < .05 (*p < .05, ***p < .001, ****p < .0001,
ns; p > .05).

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ÖZTÜRK et al.
impair bone formation and contribute to bone loss, osteoporosis, and
age- related bone fragility (Chen et al., 2015; Cicek & Cakmak, 2018).
Conversely, scavenging of ROS has been demonstrated to inhibit
osteoclastogenesis and promote bone regeneration, suggesting a
potential therapeutic approach for mitigating the adverse effects of
ROS on bone health (Dou et al., 2016). Previous studies have estab-
lished the significant impact of ROS and oxidative stress on bone
health, fracture healing, and bone remodeling processes. Therefore,
a potential correlation between the detrimental impacts of tartra-
zine on diminished bone quality and reduced bone fracture resis-
tance might be linked to ROS and oxidative stress mechanisms.
5 | CONCLUSION
Taking all into consideration, tartrazine was administered orally to
pregnant rats during gestation, which resulted in various morpho-
logical, microstructural, and biomechanical changes in the organs
and bones of the one- month- old offspring. The changes in several
organs are consistent with previous studies and were supported
by histological and real- time PCR parameters. Moreover, our study
demonstrated the effects of tartrazine on bone tissue of the one-
month- old offspring exposed to tartrazine through placental pas-
sage. This study furnishes substantiation for the clinical prevention
and treatment of developmental delays or diseases that may occur
due to the placental passage of tartrazine and its effects on offspring.
Yet, additional research is still necessary to thoroughly examine the
potential mechanism link between tartrazine and reduced bone
quality and fracture resistance. Therefore, as a future perspective,
it is crucial to comprehensively evaluate the effects of tartrazine ex-
posure both during pregnancy and in newborns post- exposure using
various methods. Additionally, investigating the long- term effects of
low- and moderate- dose, chronic tartrazine intake on bone health
and overall development could provide valuable insights.
AUTHORCONTRIBUTIONS
Osman Öztürk: Validation (equal); writing – original draft (equal).
Yusuf Dikici: Data curati on (equal). Öznur Gür: Data curation (eq ual).
Mert Ocak: Data curation (equal); formal analysis (equal). Züleyha
Doğanyiğit: Data curation (equal); formal analysis (equal). AslıOkan:
Data curation (equal); formal analysis (equal). Evrim Suna Arıkan
Söylemez: Formal analysis (equal); methodology (equal). Şükrü
Ateş: Conceptualization (equal); methodology (equal). Sümeyye
Uçar: Conceptualization (equal); methodology (equal). Mustafa
Unal: Data curation (equal); formal analysis (equal). SeherYılmaz:
Conceptualization (equal); Formal analysis (equal); Methodology
(equal); writing – review and editing (equal).
ACKNOWLEDGMENTS
There is no funding for this research.
CONFLICT OF INTEREST STATEMENT
The authors declare that they have no conflict of interest.
DATAAVAI L A BIL IT Y STATE M EN T
The data that support the findings of this study are available on re-
ques t from the co rre spo nding auth or. Th e dat a are not publ icl y av ail-
able due to privacy or ethical restrictions.
FIGURE 7 (a) A three- point bending
test conducted on femurs, accompanied
by the corresponding typical force–
displacement curve. (b) Both structural
and material- level biomechanical
properties of femurs exhibited significant
reductions in the tartrazine- exposed
groups in comparison to the control
groups (p < .05).

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ÖZTÜRK et al .
ETHICALAPPROVAL
It was carried out in accordance with the decision numbered 24/008
taken from the Erciyes University Animal Experiments Local Ethics
Committee.
ORCID
Osman Öztürk https://orcid.org/0000-0003-1156-7419
Yusuf Dikici https://orcid.org/0000-0002-7061-2550
Öznur Gür https://orcid.org/0000-0003-0175-9433
Mert Ocak https://orcid.org/0000-0001-6832-6208
Züleyha Doğanyiğit https://orcid.org/0000-0002-6980-3384
Aslı Okan https://orcid.org/0000-0001-8152-7338
Evrim Suna Arıkan Söylemez https://orcid.
org/0000-0002-8550-793X
Şükrü Ateş https://orcid.org/0000-0001-7096-2481
Sümeyye Uçar https://orcid.org/0000-0003-3378-3745
Mustafa Unal https://orcid.org/0000-0002-9518-8952
Seher Yılmaz https://orcid.org/0000-0003-4551-995X
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M., Doğanyiğit, Z., Okan, A., Arıkan Söylemez, E. S., Ateş, Ş.,
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