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RNA-seq analysis reveals transcriptional disruption

induced by combined cisplatin and TiO₂

nanoparticles in Drosophila melanogaster

Cristiano Nascimento Costa

Rio de Janeiro Federal Institute of Education, Science and Technology

Luciane Prioli Ciapina

Laboratório Nacional de Computação Cientíﬁca

Ana Cristina Bahia Nascimento

Federal University of Rio de Janeiro

Valdir Braz da Silva Neto

Federal University of Rio de Janeiro

Fabricio Alves Barbosa da Silva

Oswaldo Cruz Foundation

Francisco Lopes

Federal University of Rio de Janeiro

Research Article

Keywords:

Posted Date:

June 19th, 2026

DOI:

https://doi.org/10.21203/rs.3.rs-9987753/v1

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This work is licensed under a Creative Commons Attribution 4.0 International License.

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Additional Declarations:

No competing interests reported.

DOI:10.21203/rs.3.rs-9987753/v1

RNA-seq analysis reveals transcriptional disruption induced by

combined cisplatin and TiO

₂

nanoparticles in Drosophila

melanogaster

Costa, C. N.

1, 2

, Ciapina, L. P.

, Bahia, A. C.

, Braz, V.

, Da Silva, F. A. B.

5 *

, Lopes.

1 *

–

Universidade Federal do Rio de Janeiro, UFRJ

–

Campus Duque de Caxias Professor

Geraldo Cidade; Duque de Caxias, Brazil.

–

Instituto Federal do Rio de Janeiro, IFRJ

–

Campus Rio de Janeiro.

–

Laboratório Nacional de Computação Científica, LNCC

–

Instituto de Biofísica Carlos Chagas Filho, IBCCF - UFRJ

–

Fundação Oswaldo Cruz, FIOCRUZ

* These two authors are the last co-authors.

Corresponding author at: UFRJ - Campus Duque de Caxias Professor Geraldo

Cidade, Duque de Caxias, RJ, Brazil. E-mail address: flopes@ufrj.br. (Lopes. F).

ABSTRACT

Cisplatin, the first metal-based chemotherapeutic drug, remains widely used despite

its toxicity and the emergence of drug resistance. Combining cisplatin with nanoparticles

has been proposed to improve its therapeutic profile, although most studies rely on

in vitro

models. Here, we investigated the

in vivo

transcriptional effects of cisplatin (50 μg/ml) and

titanium dioxide nanoparticles (TiO

₂

NPs; 50 μg/ml), alone and in combination, in

Drosophila melanogaster

. Using RNA-seq and bioinformatic analyses, we assessed

differential gene expression across treatments. Flies exposed to cisplatin or TiO

₂

NPs

alone exhibited modulation of genes associated with xenobiotic metabolism and

detoxification. In contrast, combined exposure resulted in a markedly reduced number of

upregulated differentially expressed genes (DEGs):

CG10013

aqz, CG5568

, and

CG3213

, which are related to cell division and DNA/RNA metabolism. Also, we observed

that the combined exposure promotes the downregulation of DEGs involved in xenobiotic

metabolism, detoxification, collapse of the innate immune system, and reproductive

impairment and fertility. Mortality rates were not significantly affected in any group. These

results suggest that co-exposure to cisplatin and TiO

₂

nanoparticles induces a global

transcriptional suppression, potentially impairing essential cellular processes.

INTRODUCTION

Cisplatin

the

commercial

name

for

the

compound

(SP-4-2)-

diamminedichloridoplatinum (II), the first metal-based anticancer drug widely used to treat

several cancers (Ghosh, 2019; Mohammadi; Li; Ewing, 2018; Zhang

et al

., 2021). Multiple

in vitro

and

in vivo

studies have shown its mechanism of action, which involves the

generation of DNA lesions. These lesions trigger several signal transduction pathways

that lead to inhibition of replication and transcription, cell cycle arrest, DNA repair, and

apoptosis. DNA damage results from cisplatin's interaction with purine bases in DNA,

generating interstrand adducts (G-G, 60%; A-G, 20%) and DNA-protein interactions

(Allgayer

et al

., 2019).

A genotoxic study using the Comet assay indicates that, in

Drosophila

melanogaster

, detected DNA damage contains higher levels of G

–

G cross-links than G

monoadducts, thereby contributing to the induction of DNA strand breaks by blocking DNA

replication and repair. (García Sar

et al.

, 2012). Cisplatin is associated with side effects

such as nephrotoxicity and ototoxicity in humans (Karasawa; Steyger, 2015), as well as

chemotherapy-induced peripheral neuropathy (CIPN) in mammals and non-mammals,

such as

D. melanogaster

(Cirrincione; Rieger, 2020).

Besides its side effects, cisplatin is still largely used as a chemotherapeutic drug. To

reduce cisplatin's side effects, research has examined the use of different carriers in

combination with cisplatin. In this context, the combined use of 50 µg/ml TiO

nanoparticles and 10 µg/ml cisplatin improved chemotherapy response

in vitro

in the F10

melanoma model and

in vivo

in C57BL/6 mice, likely through the crucial role of

nanoparticle-induced autophagy in sensitizing F10 melanoma cells (ADIBZADEH

et al.

2021).

Although largely used in food, pharmaceutical, and cosmetic industries, titanium

dioxide nanoparticles (NPTiO

) have

in vivo

side effects that are still unconfirmed. An

vivo

test using the bivalve

Mytilus galloprovincialis

exposed to different concentrations of

NPTiO

showed altered lysosomal and oxidative stress biomarkers, along with reduced

transcription levels of immune-related antioxidant genes (Barmo

et al

., 2013). Similarly,

mice orally treated with different concentrations of NPTiO

, showed apoptotic DNA

fragmentation and mutations in p53 exons, which persisted for up to one or two weeks

post-treatment. In addition, these animals exhibited increased levels of malondialdehyde

and nitric oxide, decreased glutathione levels, and reduced catalase activity in a dose-

and time-dependent manner (Mohamed, 2015).

NPTiO

administration is directly associated with persistent inflammation, apoptosis,

oxidative stress, and the induction of chronic gastritis (Mohamed, 2015). It also directly

binds to proteins involved in cellular functions, thereby generating primary genotoxic

effects. The main mechanism of this NPTiO

genotoxic effect was attributed to reactive

oxygen species (ROS) generated by increased H

, singlet oxygen, and lipid peroxide

levels, which reduce glutathione (GSH) and antioxidant levels (Wani, Shadab, 2020).

Also, a study that examined 34 robust datasets did not support a direct DNA-damaging

mechanism for TiO

in nano- or microform (Kirkland et al., 2022).

Drosophila melanogaster, commonly known as the fruit fly, has been used as a

model organism for

in vivo

studies to elucidate many human diseases, like cancer

(Mirzoyan et al., 2019), substance toxicity (Groen

et a

l., 2022; Ong

et al

., 2015), and

also nociception (He

et a

l., 2022).

Here, we investigate the effects of

in vivo

isolated and combined administration of

cisplatin (50μg/ml) and TiO

nanoparticles (50μg/ml) in D. melanogaster via oral

administration. Furthermore, we analyzed gene modulation by identifying differentially

expressed genes (DEGs) from RNA-seq data. Downstream analysis was also performed

to verify the ontology terms associated with the different treatments in

D. melanogaster

Our data showed a specific expression pattern in flies treated simultaneously with cisplatin

and TiO2 nanoparticles, characterized by intermediate transcript levels and a minimal

number of upregulated DEGs.

MATERIALS AND METHODS

Drosophila melanogaster

maintenance

Drosophila melanogaster

strain Oregon (RRID:BDSC.5) was obtained from the

Bloomington Drosophila Stock Center, Indiana University. The culture was performed in

a standard cornmeal-yeast-agar medium (BRENT; OSTER, 1974) and maintained in an

incubator at 25 °C and humidity between 70% and 80%, with a 12-hour light cycle.

Treatments

1-to-4-day-old

D. melanogaster

strain Oregon flies were divided into four groups, each

with 30 individuals. Each group contains a 4:1 female/male ratio. The control group and

all treatment groups performed in biological triplicate. The control group was treated with

a standard medium enriched with a mixture of yeast, water, and a green food coloring

dye.

Treatment groups were treated with the same culture medium but with the following

aqueous solutions instead of water: CIS group

–

aqueous cisplatin solution (50µg/ml);

NPTIO2 group - aqueous NPTiO

solution (50µg/ml); CIS/NPTIO2 group

–

cisplatin

aqueous solution (50µg/ml) and NPTiO

aqueous solution (50µg/ml) simultaneously. Flies

were kept in this condition for 3 (three) days.

Only female flies were used for the RNA-Seq experiment.

3. Preparation of Used Solutions

NPTiO

water solution was prepared using 50mg/ml of TiO

(Sigma-Aldrich, Cat.

No.: 718467; nano powder, 21 nm primary particle size (TEM), ≥99.5% trace metals

basis), which was heated in an oven (200ºC) for 30 minutes. Then, TiO

was solubilized

in deionized water by sonication (25ºC, 20 minutes). Cisplatin (Cat. No.: PHR1624)

obtained from Merck was dissolved in Milli-

Q water (18.2 MΩ cm distilled deionized water

obtained from a Milli-Q system, Millipore, Bedford, MA, USA), forming a 50mg/ml cisplatin

solution.

4. Survival Analysis

All Drosophila survivors are used in survival analysis. Afterward, a phenotypic

analysis was conducted using a stereoscope to assess any phenotypic alterations

resulting from the treatments. The Kaplan-Meier then calculated the survival rate, followed

by the Log-rank (Mantel-Cox) test. Survival rate was performed in R Studio using the

“survival package”.

5. RNA isolation

We used two female flies from each of the treated groups (CIS, NPTIO2, and

CIS/NPTIO2) and the control group for RNA isolation. Flies were placed in 1.5 ml RNase-

free plastic tubes containing 500 µl of Trizol

™

reagent (ThermoFisher

™

Scientific,

Waltham, Massachusetts, USA) and macerated. Total RNA extraction was made

according to the manufacturer’s instructions. At the end of the extraction, the pellet

containing RNA was resuspended in 20 mL of water.

To avoid DNA contamination, we used the RNase-Free DNase Set (QIAGEN, Cat.

No.: 79254) according to the manufacturer's instructions. RNA integrity was analyzed

using agarose gel visualized in Molecular Imager

Gel Doctm XR+ Imaging system (Bio-

Rad Laboratories, Inc.) and quantified with NanoDrop 2000 spectrophotometer

(ThermoFisher

™

Scientific) and a Qubit 3.0 Fluorometer (ThermoFisher

™

Scientific).

6. RNA-seq

RNA-Seq library preparation and sequencing were performed at Computational

Genomics Unity Darcy Fontoura de Almeida (UGCDFA) of the National Laboratory of

Scientific Computation (LNCC) (Petrópolis, RJ, Brazil). The mRNA sequencing libraries

were constructed using the Illumina Stranded mRNA Prep Ligation kit (Illumina, San

Diego, CA, USA), according to the manufacturer’s protocol. The quality of libraries was

verified using the Agilent TapeStation 4200 System (Agilent Technologies, USA)

according to the manu

facturer’s instructions. Sequencing was performed on an Illumina

NextSeq 500 platform (Illumina, San Diego, CA, USA) using the NextSeq 500/550 high-

output kit v2.5 (150 cycles) (Illumina, USA), generating 2 × 75

-bp reads.

7. Analysis of RNA-Seq data

The quality of the raw sequence data was assessed using FastQC

(http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Adapter sequences and low-

quality regions were removed by trimming the first five nucleotides from the 5’ end and
the last 40 nucleotides from the 3’ end of each replicate. Reads shorter than 20

nucleotides after trimming were removed using the Trimmomatic tool (Bolger; Lohse;

Usadel, 2014). The remaining readings were aligned to the reference genome of

Drosophila melanogaster

ASM2977511v1, date: April 17

, 2023 (BioProject, NCBI), using

the BWA aligner (Li; Durbin, 2009). The alignment quality was evaluated using Samtools

(Li et al., 2009), and the number of reads mapped to each gene was quantified using HT-

seq Count tool (Anders; Pyl; Huber, 2015).

8. Statistical analysis

DESeq2 (Love; Huber; and Anders, 2014) was used for differential gene expression

analysis. Differentially expressed genes (DEGs) were identified using a log2 fold change

(log2FC) cutoff > 1 and an adjusted p-value (padj) < 0.05. All treated groups were

compared against the control group.

9. Downstream analysis

Functional enrichment analysis of DEGs was performed using the databases:

FlyBase (Öztürk-Çolak

et al

., 2024), FlyEnrichr (Kuleshov

et al.

, 2019), and Gene

Ontology (GO) (The Gene Ontology Consortium, 2019).

10. Data availability

The data discussed in this publication have been deposited in NCBI's Gene

Expression Omnibus (Edgar

et al.

, 2002) and are accessible through GEO Series

accession number GSE334628 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=

GSE334628).

All

codes

used

this

paper

are

available

at:

https://github.com/users/CristianoNCosta/projects/1

RESULTS AND

DISCUSSION

Data analysis

To evaluate the individual and combined

in vivo

effects of cisplatin (50 μg/mL) and

TiO

nanoparticles (50 μg/mL),

Drosophila melanogaster

mortality rates were measured

as described in the Materials and Methods section. As illustrated in Figure 1, no significant

variation was observed among the evaluated groups (p = 0.76).

Figure 1: Kaplan

–

Meier survival curves of Drosophila melanogaster exposed to different treatments

Control, cisplatin (50

g/ml CIS), titanium dioxide nanoparticles (50

g/ml NPTIO2), and the combination CIS/NPTIO2

(50

g/ml/50

g/ml). Survival rates were similar across all experimental groups (p = 0.76). The Log-rank (Mantel

–

Cox)

test showed no statistically significant differences among the groups (Events: 18; p-value: 0.75347), indicating that none
of the treatments significantly affected fly survival over the 3-day observation period.

To evaluate sample expression variability following DESeq2, we conducted a

Principal Component Analysis (PCA) using the BiocManager package. Within this

framework, the standard PCA executed by the DESeq2 package applies a regularized

logarithm (rlog) transformation for data normalization. PCA is a widely accepted method

for variance analysis. As shown in Figure 2, this analysis suggests that CIS/NPTIO22

represents an outlier sample.

Figure 2

PCA plot of RNA-seq data (rlog-normalized) showing sample separation by treatment.

controls (blue)

cluster on the left, while CIS (red), CIS/NPTIO2 (green), and NPTIO2 (purple) form distinct groups along PC1 (85%
variance). PC2 explains minimal variation (11%). CIS/NPTIO22 exhibits an intermediate profile between CIS and
NPTIO2.

The rlog applies a variance-stabilizing log transformation tailored to count data and

prioritizes biological interpretability. Robust Scaler is another normalization method that

standardizes data using the median and interquartile range (IQR), making it resistant to

outliers and generally applicable (not RNA-seq-specific), with a primary focus on statistical

robustness. To optimize PCA results by balancing variance stabilization and outlier

resilience, we performed a PCA analysis that combines both methods (rlog followed by

Robust scaling).

Here, we analyzed the similarity of expression patterns across the top 10,000 most

differentially expressed genes (DEGs) in each treated group after robust scale analysis,

comparing within the CONTROL group (lower p-values). Then, we performed a

quantitative analysis to verify the Euclidean distances among the replicates, as shown in

Table 1, and between CIS/NPTIO22 and all other samples. This data suggests that

CIS/NPTIO22 is an outlier.

Table 1: Euclidean Distance between CIS/NPTIO22 sample and other samples. Scale units in standard deviation

(Robust Scaler).

CIS1

CIS2

CIS3

CIS/NPTIO21

CIS/NPTIO23

4974.11

4949.75

4944.15

4950.60

4940.42

CONTROL1

CONTROL2

CONTROL3

NPTIO21

NPTIO22

NPTIO23

4919.46

4926.30

4950.49

4948.30

4943.52

4933.49

The two-dimensional PCA (Figure 3) showed that CIS/NPTIO22 is more than 4000

Standard Deviation (SD) units apart from other CIS/NPTIO2 replicates, and similar

distances are observed for all samples from other treatment groups. These data are

consistent with the outlier condition of CIS/NPTIO22, indicating that this sample should

be excluded from further analysis to maintain data integrity.

After outlier removal, PCA and quantitative analysis were repeated to assess the

Euclidean distances among the replicates. As shown in Figure 3, PC1 and PC2 explain

70% and 8% of the total variance, respectively. The distances among samples along PC2

indicate only minor biological variation within groups. Notwithstanding this, across the

PC1 axis, the most variable group is CONTROL. CONTROL3 appears more distant

(approximately 40%) from CONTROL1 and CONTROL2, though still within the range of

expected biological variability. Despite this heterogeneity, the control group. Despite this

heterogeneity, the control group remains useful for comparing transcriptional variance

across all treatment groups. The CIS group was the most homogeneous, since its

samples are closer to each other in both components.

Besides that, PC1 shows heterogeneous responses across samples within

treatment groups. NPTIO21 is separated from NPTIO22 and NPTIO23 in both PC1 and

PC2, whereas CIS/NPTIO21 is separated from CIS/NPTIO23 only in PC1. These results

demonstrate biological variation among samples within those groups, but the variation is

not sufficient to discard any of the samples. PC1 explains the largest proportion of

variance and primarily separates the treatment groups (CIS, NPTIO2, and CIS/NPTIO2)

from the control group, indicating a treatment effect.

To verify the absence of additional outliers, we computed Euclidean distances

among the replicates after removing the initial outliers. Neither the control nor the treated

group has a distance greater than 750 from other replicates, indicating no further outliers

(Supplemental material 01).

Figure 3.

Two-dimensional PCA projection of rlog-transformed count data following

quality control and outlier removal.

Samples are colored by experimental conditions:

CONTROL (blue), CIS (red), CIS/NPTIO2 (orange), and NPTIO2 (green). Variance

explained: PC1 (76%), PC2 (8%).

Taken together, Figure 3 and Table 1 demonstrate that removing the CIS/NPTIO22

outlier yields greater consistency across biological replicates and supports robust

inference about treatment effects, enabling more reliable downstream comparisons and

interpretation.

Gene Expression and Downstream Analysis.

Expanding upon the above analysis, we next examined the global patterns of gene

expression and differential regulation elicited by each treatment group.

Volcano plots presented in Figure 4 compare total transcript counts across

treatments, showing the distribution of DEGs (fold change (log2fc)> |1.0| and p-value <

0.05) for each treated group (CIS, NPTIO2, and CIS/NPTIO2) relative to the control.

The total transcript counts by each control or treatment group are indicated in Figure

5. Comparing the treatment groups with CONTROL, our data show that both CIS and

CIS/NPTIO2 have higher median total transcript counts than CONTROL. However, the

NPTIO2 treatment alone appears to have a lower total transcript count compared with

other treatment groups. As indicated in Figure 5, the application of these treatments

(particularly those involving CIS) increased transcriptional activity relative to the baseline

of the control group.

Although the CIS/NPTIO2 group presented a lower number of upregulated and

downregulated DEGs, the position of its median and interquartile range indicates that the

combination of cisplatin (50 μg/ml) and TiO

nanoparticles (50 μg/ml) is effective in

keeping the

Drosophila’s

global transcription machinery in a state of hyperactivity.

Figure 4.

Differential gene expression analysis.

Volcano plots display the relationship between statistical

significance (-log

₁₀

p-value) and the magnitude of change (log

₂

fold change) for all measured genes. (a) CIS

treatment versus CONTROL, (b) NPTIO2 treatment versus CONTROL, (c) CIS/NPTIO2 treatment versus
CONTROL. DEGS with differentiated expression (fold change (log2fc > |1.0| and p-Value <0.05): Significant (

•

Not significant (•).

Figure 5. Total Transcript Counts by Treatment Group:

Box plots for each of the four treatment groups

(CIS, CIS/NPTIO2, CONTROL, and NPTIO2). The line within each box indicates the median, and the box
represents the interquartile range (IQR). Black dots represent individual sample data points. Total transcript
counts are highest in the CIS and CIS/NPTIO2 treatment groups, while the CONTROL group shows the
lowest counts.

After confirming the statistical data and removing outliers, we began analyzing the

DEGs in each group. Figure 6 displays a Venn diagram showing the numbers of uniquely

upregulated and downregulated DEGs per group, alongside the shared DEGs found in

two or more groups. We detected 28 upregulated and 890 downregulated DEGS in the

CIS group, 17 upregulated and 400 downregulated DEGs in the NPTIO2 group, and only

4 upregulated and 61 downregulated DEGs in the CIS/NPTIO2 group. The number of

downregulated DEGs in the CIS/NPTIO2 group indicates that both cisplatin and TiO

have

a repressor effect on gene expression in this group.

Two DEGs are upregulated across all groups (

CG10013

and

aqz

). The CIS and

NPTIO2 groups have 4 upregulated DEGs (

CG11263, CG18088, Tango6, inaF-D

) and

one long non-coding RNA (lncRNA: CR43836). One upregulated DEG is present in the

CIS and CIS/NPTIO2 groups (

CG5568

) and in the NPTIO2 and CIS/NPTIO2 groups

(

CG3213

Conversely, treatment conditions yield distinct profiles of downregulated DEGs

across groups. Approximately 19,000 genes were differentially expressed, of which only

1,056 were downregulated DEGs. Among these, 22 DEGs were consistently

downregulated across all treated groups. The CIS group had the highest number of

downregulated DEGs (890), whereas the CIS/NPTIO2 group had only 61.

The reduced number of up- and downregulated DEGs, specifically in the

CIS/NPTIO2 group, suggests a possible synergistic effect of the combined administration

of cisplatin and TiO

nanoparticles in

Drosophila

. The observed results are likely due to

reduced cell signaling following the joint administration of these substances in

Drosophila

as previously demonstrated (Adibzadeh

et al.

2021).

Figure 6

: Venn diagram relative to the number of DEGs obtained in (a) upregulated DEGs and (b)

downregulated DEGs in the challenged groups.

(a)

(b)

Figure 7 illustrates the downstream analysis of upregulated DEGs detected in

Drosophila

treated with 50μg/ml

cisplatin, which, according to FlyEnrichr (Kuleshov

et al.

2019) shows no significant KEGG pathway, but presents 31 upregulated DEGs and 3 long

non-coding RNA. The upregulated DEGs in the CIS group possess biological process GO

terms related to stimulus response, development, biogenesis, DNA metabolism, and

immune response. Molecular function GO terms correspond to enzyme, metal ion, RNA,

and carbohydrate binding. Additionally, cellular component GO terms for these DEGs

relate to the membrane, cell periphery, cytosol, nucleus, and others, as compiled in

Supplementary Table 1. This data indicates that

Drosophila

treated with 50 µg/ml cisplatin

upregulates DEGs related to metabolic responses to stimuli and DNA repair machinery.

It is distinct from previous data, which identifies DEGs upregulated and downregulated 50

μg/mL

and 100

μg/mL

cisplatin concentrations (Mombach

et al.

, 2022).

Figure 7 -

GO enrichment analysis of upregulated genes under CIS treatment

: (

■

) Biological Process, (

■)

Cellular

Component, and (

■

) Molecular Function. Full results are in Supplementary Table 1. Data source GO Summary Ribbon

(Fly Base).

Flies treated with 50 μg/mL of NPTiO

(NPTiO2 group) exhibited upregulation of

16 differentially expressed genes (DEGs) and one long non-coding RNA. Additionally, two

DEGs were upregulated across all treatment groups, four DEGs were shared with the CIS

group, and one DEG was upregulated in both the CIS and NPTiO2 groups. These DEGs

are associated with 10 biological process GO terms (response to stimulus, gene

expression, protein metabolism, development, signaling, other biological processes,

transport/localization, cell cycle/proliferation, immune system, and DNA metabolism), 6

molecular function GO terms (small molecule binding, RNA binding, transport, enzyme,

and receptor binding), and 9 cellular component GO terms (macromolecular binding,

nucleus, membrane, chromosome, and endomembrane system) (Figure 8). While

Jovanović, Cvetković, and Mitrović (2016) demonstrated that gene expression is

unaffected by NPTiO2 uptake, they observed activation of DEGs related to stimulus

response and transport/cell localization; our data support this observation.

Figure 8:

GO enrichment analysis of upregulated genes under NPTIO2 treatment

: (■) Biological Process,

(

■

) Cellular Component, and (

■

) Molecular Function. Full results are in Supplementary Table 1. Data source GO

Summary Ribbon (Fly Base).

Finally, four upregulated DEGs are observed in the CIS/NPTIO2 group (flies treated

with 50μg/ml of

cisplatin

and 50μg/ml of NPTiO

concomitantly) as indicated in Table 2.

Figure 8 also indicates that NPTiO

upregulated genes are related to cytoskeleton and

protein metabolism genes, which are normally repressed in flies treated only with cisplatin,

but are active in flies treated with nanoparticles in general (Adibzadeh

et al.

, 2021;

González-López

et al.

, 2020; Mohammadi; Li; Ewing, 2018). As these genes are related

to endocytosis, this suggests a possible increase in cisplatin incorporation due to the

effect of NPTiO

when administered together.

Table 2: Gene Ontology (GO) enrichment analysis of CIS/NPTIO2-upregulated genes

GO Category

Subcategory

Nº Genes

Cellular Component

cytoskeleton

nucleus

GO Category

Subcategory

Nº Genes

Biological Process

fatty-acyl-CoA biosynthetic process

long-chain fatty acid metabolic process

biological_process

Molecular Function

enzyme

Table 2

: DEGs from CIS/NPTIO2 group classified using GO Summary Ribbon (Fly Base). Only the most specific

terms are shown. Full data available in Supplementary Table 1.

We identified two genes upregulated across all three treatment groups:

CG10013

and

aqz

CG10013

encodes a ubiquitin-conjugating enzyme E2-binding protein that

accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes and transfers it to

substrates, generally promoting their degradation by the proteasome. CG10013

contributes to genome structural dynamics in

Drosophila

evolution as a key element in

the evolution of the Homeobox complex (HOX-C) (Negre; Ruiz, 2007). Furthermore,

CG10013

has potential involvement in pigmentation variation, alongside other genes

known to be involved in melanin production, such as

ebony, tan, yellow,

and

black

(Bastide

et al.

, 2016). The gene

aqz

is predicted to be active in the nucleus and codes for

the Aaquetzalli protein isoform A.

aqz

is orthologous to human

PNRC1

(proline-rich

nuclear receptor coactivator 1) and

PNRC2

(proline-rich nuclear receptor coactivator 2)

and is related to neural tissue formation and nervous cells polarity (Mendoza-Ortíz;

Murillo-Maldonado; Riesgo-Escovar, 2018). Our data indicate that together,

CG10013

and

aqz

play a role in responses to oxidative stress and other conditions affecting the cell,

as evidenced by their upregulation across all treated groups.

The upregulation of

CG5568

observed in flies from both the CIS and CIS/NPTIO2

groups is primarily elicited by cisplatin exposure. Functionally, this gene encodes a zinc-

finger DNA-binding protein involved in the negative regulation of the meiotic cell cycle and

eye development (Morrison

et al.

, 2006). It is also highly expressed in female germline

cells and implicated in the fatty-acyl-CoA biosynthetic process (Li

et al.

, 2022). Our

findings highlight that the increased expression of CG5568 is expected as a specific

response to cisplatin treatment.

CG3213

is upregulated in flies from both NPTIO2 and CIS/NPTIO2 groups; this gene

is involved in ribosomal and protein translation (Pospisilik

et al.

, 2010). Also,

CG3213

was

identified in a genome-wide screen for obesity-related genes in Drosophila, and a single-

nucleotide polymorphism (SNP) in

CG3213

is associated with variation in starvation

resistance (Mackay

et al.

, 2012). A screening performed in adult flies, including both

sexes, demonstrates that triglyceride levels, a direct measure of adiposity, were

determined in adult flies following RNAi induction. Inhibition of

CG3213

in muscle cells

was shown to regulate triglyceride levels (Garlovsky; Sandler; Karr, 2022). Yet

CG3213

is associated with distinct processes in the germline stem cell lineage (Matsuoka et al.,

2017). Upregulation in DEGs related to fatty-acid and lipid metabolism in flies treated with

TiO

nanoparticles (NPTiO

) is expected, as mentioned in Figure 8.

DEGs upregulated by the CIS/NPTIO2 group alone or combinedly in one or the other

two treated groups are related to biological processes described in Table 2, specifically

those related to fatty-acid and lipid metabolism, which strongly suggests a possible

increase in the incorporation of cisplatin due to the endocytosis mediated by the NPTiO

joint administration.

Drosophila

from both CIS and NPTIO2 groups have 3 upregulated DEGs in common

(

CG11263, CG18088

and

InaF-D

) and an upregulated long non-coding RNA

(lncRNA:CR43836). Recent data suggest that long non-coding RNAs (lncRNAs) may

have important regulatory functions in the genome (Zhong

et al.

, 2020). Specifically

regarding lncRNA CR43836, data show that this lncRNA is expressed in many embryonic

tissues, larval visceral muscle cells, and adult females (Matthews

et al.

, 2015).

CG11263

is an RNA-binding protein related to lipid binding, cilium assembly, and intracellular

transport, expressed in

Drosophila

embryos, prepupa and pupa stages, and in adult

females (ADAMS, 2000). Upregulation of

CG11263

could explain the presence of

lncRNAs in treated flies in these groups, given its role in transcriptional regulation.

CG18088

is associated with

Drosophila’s

immune response to

Wolbachia

. and affects

transduction-related genes.

InaF-D

binds to the transient receptor potential (TRP) calcium

channel and affects metal absorption (Chen; Montell, 2020). The last DEGs are also

related to immune response and metal response and are expected to be upregulated in

flies treated with those metal compounds (López-Varea

et al.

, 2021).

All flies were kept for 3 days and fed food supplemented with wet yeast paste

containing cisplatin, NPTiO

, or both substances, without prior starvation. This can

positively affect cell proliferation in flies, regardless of group (control or treated). Several

studies demonstrate that protein intake affects oogenesis in the female ovary, so the

observation of upregulated DEGs related to this function is expected (Ables

et al.

, 2016;

Ables, Laws, Drummond

‐

Barbosa, 2012). No upregulated DEGs related to oogenesis

were detected in this study.

Previous studies have demonstrated that mated females can store sperm transcripts

in their spermatheca, and RNAs related to sperm from many genes, including those of

accessory gland and seminal fluid proteins, can be detected (Cridland; Begun, 2023;

Prokupek

et al.

, 2008). Our treatment conditions, feeding time, food supplementation

provided to flies, use of mated females, and the pipeline and sequencing technologies

used could explain the difference between our results and those of the previous study in

flies treated with the same cisplatin concentration (Mombach

et al.

, 2022). Also, it could

explain the identification of DEGs primarily related to

Drosophila

males but also expressed

in females in our data.

CIS and NPTIO2 groups presented a response whose enrichment has the GO terms

(Figures 7 and 8) related to response to stimulus, gene expression, protein metabolism

and development (BP); and enzyme, nucleic acid binding and small molecule binding

(MF); DEGs in these groups are located in different cellular components but enrichment

GO terms related to the membrane, endomembrane system, and macromolecular

complex has found in both. Our data are partially consistent with a previous study

indicating that, regardless of the treatment to which Drosophila are subjected, upregulated

genes have GO terms related to stimulus response and include genes encoding

lysozymes, cytochrome, and mitochondrial components (BROWN

et al

., 2014).

Our data show upregulated DEGs related to different Response to stimulus GO

terms. According to GO Summary Ribbons (FlyBase (FB2025_03, released July 24,

2025)), the CIS group has six up-regulated DEGs with Response to stimulus GO Term:

FANCI, St4, Ir7c, PPO1, out,

and

CG10738

. A more detailed analysis of the FlyEnrichr

database indicates that this set of DEGs is associated with the following GO biological

processes, including defense response to bacteria, oogenesis, regulation of cell

proliferation, and wound healing, among others (Supplementary Table 2). The molecular

functions of this set of DEGs are related to sulfotransferase activity, DNA polymerase

binding, and hydrolase (Supplementary Table 2).

CIS group flies also present three upregulated lncRNA (lncRNA:CR43836,

lncRNA:CR44707, lncRNA:CR45052). lncRNA:CR43836, as mentioned before, and all

knowledge regarding these lncRNAs is related to regulation in transcription. Our data is

according to the state of the art, which indicates that cisplatin treatment induces lncRNAs-

mediated responses ((Brown

et al

., 2014; Zhang

et al.

, 2020).

The CIS group also has DEGS related to development:

CG9503, dec, Cad88C,

Cad74A,

and

out

. The FlyEnrichr database shows that this set of DEGS is associated with

the following GO biological processes: cell-cell adhesion, female germline sex

determination, regulation of DNA endoreduplication, and ecdysteroid metabolic process,

among others (Supplementary Table 1). These molecular functions are related to

hydrolase activity and ionic binding (Supplementary Table 2). The combined

superexpression of all these genes suggests that the organism is undergoing an intense

phase of development, differentiation, and tissue reorganization. They appear to be

working together to coordinate the complex morphological changes that occur in the

Drosophila

body in response to cisplatin.

In the NPTIO2 group, we found upregulation in

CG1503, l(2)03659

, and

CG44838

Its upregulation suggests Drosophila treated with 50μg/ml of TiO

needs to activate

transport machinery to metabolize TiO

. In case,

CG44838

is a transcription factor known

for signal transduction and nervous system development (Olson

et al.

, 2019) and activate

CG1503

which is a protein regulator having hydrolase activity and are related to

decreased carbohydrate metabolism (Hu

et al.

, 2021), finally

l(2)03659

which is related

to response to xenobiotics or metabolites mitochondrial stress-relief mechanism

associated with multidrug resistance (Yeh

et al.,

2017) due to activation in transmembrane

transport mechanism.

CG11263, tut,

and

CG18088

DEGs were upregulated by NPTiO

. These DEGs are

associated with the negative regulation of gene expression and the piRNA metabolic

process.

CG11263

is co-expressed and functions as part of the transcription machinery

that generates piRNA precursors from heterochromatin while maintaining suppression of

transposon-encoded promoters and enhancers. This is also related to the viability of

pupae and adult females (Matthews

et al.

, 2015).

CG10013

and

ord

are co-expressed

and promote normal formation and maintenance of the synaptonemal complex and

centromere clustering (Das

et al.

, 2016). Also,

ord

is essential for proper maintenance of

sister-chromatid cohesion in both male and female meiosis. Yet, mutations in this gene

cause premature separation of sister chromatids in meiosis I, random segregation in both

meiotic divisions, and reduced recombination in female meiosis (Miyazaki; Orr-Weaver,

1990).

D. melanogaster

challenged with nanomaterials develops acute effects related to

changes in pigmentation, survival rate, life span, and genotoxicity (Zhang

et al

., 2023).

Relative to NPTiO

, a study using

D.melanogaster

chronically exposed to suspensions

containing 5, 10, 15, and 20 mg/kg NPTiO

from the embryonic period of the P0 to F3 in

the adult stage, demonstrates a decrease in the total crawling distance of larvae and the

total movement distance of adult males in F3. Results obtained in this study suggest

disruption in the Impaired Neuromuscular Junction (NMJ) due to an increased expression

level of genes related to response to Lithium-ion Response (

List

) and neuron cellular

homeostasis (

Gba1a

and

hll

), and a decrease in gene expression levels of thermosensory

behavior gene (

Cyp6a17

) and axon target recognition gene (

frac

) (ZHANG

et al.

, 2023).

Our data, which are based on prolonged but not chronic exposition, corroborate these

observations regarding the reduction in the expression of

frac

and

Cyp6a17

Based on our observations

aqz

and

CG10013

, which are DEGs upregulated by all

treated groups (CIS, NPTIO2 and CIS/NPTIO2), should have a significant role in the fly

response to cisplatin and NPTiO

. Also, the upregulation of these DEGs across all treated

groups indicates that ribosomal production and metabolism are affected by these

treatments.

Of particular interest is the observation that the 61 downregulated DEGs in

CIS/NPTIO2 group, mostly are related to molecular functions (MF) of hydrolase, serine

and serine-peptidase activity; the repression of these MFs may lead to a drastic defects

in embryonic development, collapse of the innate immune system, reproductive

impairment and fertility, loss of resistance to insecticides and xenobiotics, dysregulation

of lipid metabolism and phagocytic degradation (Kumar

et al.

, 2021; Zhang

et al.

, 2020).

Also, this could be related to high embryonic lethality, pupation failure, and severe

immunodeficiency in flies from this group (Marra

et al.

, 2021; Mulinari; Häcker; Castillejo-

López, 2006).

In conclusion, our data suggest that metabolic alterations occur in

Drosophila

within

the CIS/NPTIO2 group, particularly affecting cell-division metabolism, which is strongly

affected by the combined use of cisplatin and NPTiO2. Although

Drosophila

in this group

show no statistically significant difference in survival, a strong quiescence in cell-division

metabolism is observed. Therefore, the combined administration of cisplatin and TiO

₂

nanoparticles may be useful in treating cancers for which cisplatin alone is ineffective.

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