Synergistic effects of combined exposure to hydroquinone and nitrofurantoin in T24 bladder cells: an in vitro toxicology study with implications for risk assessment

Name	Concentration (μg/mL)	Description
Nitrofurantoin^*	167 (0.16 % DMSO)	prophylactic dose (100 mg of nitrofurantoin consumed orally)
Nitrofurantoin^*	330 (0.3 % DMSO)	minimal therapeutic dose (50 mg, 4× daily – 200 mg/day)
670 (0.6 % DMSO)	maximal therapeutic dose (100 mg, 4× daily – 400 mg/day)
Hydroquinone	5; 20; 50; 320; 900	concentrations that are present in the urinary bladder after standard therapeutic application of bearberry extract (15)

* All concentrations are expressed if only 50 % of nitrofurantoin is eliminated through urine (300 mL); conversion factor 1.66 (33)

Cytotoxic (neutral red) and antioxidative/prooxidative (DCHF-DA) activity of a) nitrofurantoin, b) hydroquinone, and their mixture in unrecovered (c1) and recovered over 24 h (c2) human urinary bladder cancer T24 cells. * Statistically significant result compared to the control (100 %; one-way ANOVA followed by post hoc Tukey's HSD test; P<0.05)

Table 2

Benchmark dose (BMD) and confidence intervals BMDI (BMDL-BMDU) for hydroquinone plus nitrofurantoin mixtures and results of factorial regression analyses to determine their effect on T24 cell survival and induction of ROS after 8 h exposure

Treatment	Response	PROAST software^#				STATISTICA software^*			Activity
Treatment	Response	BMD	BMDL (μg/mL)	BMDU	Effect	Multiple R (model testing)	P-value	β₁₂	P-value
Unrecovered cells	% of survival	10.64	5.76	15.2	yes	0.64	<0.01	+2.21×10⁻⁵	0.11	/
Recovered cells (after 24 h)	% of survival	33.99	21.6	49	yes	0.72	<0.01	+9.38×10⁻⁵	<0.01	synergistic
Unrecovered cells	ROS induction	16.04	13.6	21.2	yes	0.53	<0.01	+0.0001	<0.01	synergistic
Recovered cells (after 24 h)	ROS induction	15.4	0.532	227	no	0.62	<0.01	−1.55×10⁻⁵	0.48	/

# BMD – benchmark dose; BMDL – lower 95 % confidence limit of the benchmark dose; BMDU – upper 95 % confidence limit of the benchmark dose; confirmation of the dose-response effect: BMD/BMDL <20 and BMDU/BMDL <50.

* Factorial regression model: positive (+) β12 factor – synergistic effect/activity, negative (−) β12 factor – antagonistic effect/activity (only if P<0.05)

Table 3

Formation of micronuclei (MN), nucleoplasmic bridges (NPB), and nuclear buds (NB) in T24 cells after treatment with nitrofurantoin (N), hydroquinone (HQ), and their mixture over 2 and 8 h

Sample	Time	Micronuclei							Nucleoplasmic bridges		Nuclear buds
		Total (MN)₆₀₀₀ ± SD	Mean (MN)₁₀₀₀ ± SD	Total (BN_MN)₆₀₀₀ ± SD	Mean (BN_MN)₁₀₀₀ ± SD	Distribution of BN_MN cells with			Nucleoplasmic bridges		Nuclear buds
		Total (MN)₆₀₀₀ ± SD	Mean (MN)₁₀₀₀ ± SD	Total (BN_MN)₆₀₀₀ ± SD	Mean (BN_MN)₁₀₀₀ ± SD	1 MN	2 MN	3 MN	Mean (NPB)₁₀₀₀ ± SD	Total (NPB)₆₀₀₀ ± SD	Mean (NB)₁₀₀₀ ± SD	Total (NB)₆₀₀₀ ± SD
NC₁	2 h	86	14.3±0.52	78	13±1.05	71	6	1	1.2±0.75	7	1.3±0.75	8
NC₂		75	12.5±1.03	69	11.5±0.53	63	6	0	1.5±0.84	9	0.7±0.51	4
N (167 μg/mL)		↑135^e	22.5±3.99	↑127	21.2±4.36	118	7	1	9±2.53	↑54^d,e	5±1.55	↑30^e
HQ (5 μg/mL)		↑125^e	20.8±4.02	↑110^e	18.3±2.94	96	13	1	10.3±1.51	↑62^d	5.5±1.52	↑33^e
HQ (10 μg/mL)		↑145^e	24.2±2.93	↑135	22.5±2.26	125	10	0	18.5±3.39	↑111^a,b,c	6.2±1.47	↑37^a
MIX 1		↑187^a,b,c,d	31.2±5.15	↑165^a,c	27.5±4.46	141	23	0	14.2±4.79	↑85^a,b	8.5±2.2	↑51^a,b
MIX 2		/	/	/	/	/	/	/	/	/	/	/
PC		↑138^e	23 3±3.33	↑107^e	17.8±2.21	17	17	4	7.3±2.1	↑44^d,e	3.5±0.92	↑21^d
NC₁	8 h	72	12 1±.09	68	11.3±1.37	6	6	0	1.2±0.41	7	0.8±0.05	5
NC₂		65	10.8±1.47	62	10.3±1.51	2	2	0	1±0.15	6	0.7±0.03	4
HQ (5 μg/mL)		↑167^d	27.8±2.99	↑143^d	23.8±1.17	18	18	3	30.2±2.08	↑46^a	5.5±1.05	↑33^a,c
HQ (10 μg/mL)*		↑133^{a,c, *}	22.2±3.65	↑112^{a,c, *}	18.7±2.73	13	13	4	19±1.03	↑19^{a, *}	7.3±1.97	↑44^{a,b, *}
PC		↑147^d	24.5±3.99	↑133^d	22.2±3.13	14	14	0	11.8±3.06	↑71^b,c	8.5±1.16	↑51^b,c

NC₁ – negative control, non-treated cells; N – nitrofurantoin; HQ – hydroquinone; MIX 1 – mixture of N (167 μg/mL) and HQ (5 μg/mL); MIX 2 – mixture of N (167 μg/mL) and HQ (10 μg/mL); PC – positive control (0.4 μg/mL); NC₂ – negative control, cells treated with 0.16 % DMSO used for stock of nitrofurantoin preparation.

* Expressed on a total of 3000 binucleated cells or 500 per sample. ↑ significantly higher than NC₁ (P<0.05; chi-squared test). Different superscript letters denote significant difference between groups (P<0.05; chi-squared test):

a – from PC,

b - from N,

c – from HQ (5 μg/mL),

d – from HQ (10 μg/mL),

e – from MIX 1

Table 4

Cell viability, cytostatic effect, and parameters of cell proliferation in T24 cells after treatment with nitrofurantoin, hydroquinone, and their mixture over 2 and 8 h

Sample	Time	AP (%)	Cytostatic effect		Parameters of cell proliferation
Sample	Time	AP (%)	CBPI	% cytostasis	NDI	RI (%)
NC	2 h	0.13	1.718	0	1.726	100
N (167 μg/mL)		0.17	↓1.445	27.3	↓1.447	61.94
N (330 μg/mL)		0.18	↓1.259	45.9	↓1.261	36.94
HQ (5 μg/mL)		0.15	1.695	2.3	1.699	96.82
HQ (10 μg/mL)		0.25	1.666	5.2	1.671	92.89
MIX 1		0.13	↓1.225	49.3	↓1.225	31.28
MIX 2		0.15	↓1.077	64.1	↓1.077	10.68
NC	8 h	0.17	1.558	0	1.561	100
N (167 μg/mL)		0.25	↓1.058	50.0	↓1.059	10.29
HQ (5 μg/mL)		0.28	1.449	10.9	1.453	80.51
HQ (10 μg/mL)		↑0.83^*	↓1.229	32.9	↓1.231	40.96

AP – number of cells in apoptosis (%); CBPI – Cytokinesis-block proliferation index; HQ – hydroquinone; MIX 1 – mixture of N (167 μg/mL) and HQ (5 μg/mL); MIX 2 – mixture of N (167 μg/mL) and HQ (10 μg/mL); N – nitrofurantoin; NC – negative control; NDI – nuclear division index; RI – replication index. ↓ significantly lower than NC (P<0.05; chi-squared test).

* Expressed on a total of 3000 cells

Table 5

Proteomic analysis results obtained after treatment with nitrofurantoin, hydroquinone, and their mixture over 8 h (fold change cut-off ≥1.5 and ≤0.667)

Condition	Protein name^a	Accession number^b	Alternate ID^c	Permutation test (P-value)	Fold change^d
Hydroquinone	Albumin	A0A0C4DGB6	ALB	< 0.0001	0.3
	Guanine nucleotide-binding protein G(s) subunit alpha isoforms short	A0A7I2V5R6	GNAS	0.001	0.4
	Sideroflexin-1	Q9H9B4	SFXN1	0.004	0.5
	THO complex subunit 4	E9PB61	ALYREF	< 0.0001	0.4
	CD44 antigen	P16070	CD44	< 0.0001	0.5
	Tropomyosin alpha-3 chain	A0A087WWU8	TPM3	0.003	0.5
	Zinc transporter ZIP3	F5H385	SLC39A3	0.003	0.3
	Heterogeneous nuclear ribonucleoprotein U	A0A1W2PPS1	HNRNPU	0.002	0.5
	Cathepsin B	A0A7P0NGZ6	CTSB	0.002	0.5
	Succinate–CoA ligase [ADP/GDP–forming] subunit alpha, mitochondrial	P53597	SUCLG1	0.001	0.6
	Non-histone chromosomal protein HMG–17	P05204	HMGN2	0.003	0.55
	Procollagen–lysine,2–oxoglutarate 5–dioxygenase 2	O00469	PLOD2	< 0.0001	1.5
	Synaptic vesicle membrane protein VAT–1 homolog	Q99536	VAT1	0.003	1.7
	Filamin-C OS=Homo sapiens	Q14315	FLNC	0.0009	1.7
	Mesencephalic astrocyte-derived neurotrophic factor	P55145	MANF	0.004	1.7
	Tyrosine-protein phosphatase non–receptor type 1	P18031	PTPN1	0.0003	2.3
	Delta–1–pyrroline–5–carboxylate synthase	P54886	ALDH18A1	< 0.0001	1.9
	UDP–glucose:glycoprotein glucosyltransferase 1	Q9NYU2	UGGT1	< 0.0001	2.0
	Histone H1.4	P10412	H1–4	0.002	2.4
	Lysophospholipid acyltransferase 7	Q96N66	MBOAT7	0.001	4.2
	ATP-dependent 6–phosphofructokinase, platelet type	Q01813	PFKP	0.001	4.4
	LIM domain only protein 7	F8WD26	LMO7	0.001	3.3
	MARCKS–related protein	P49006	MARCKSL1	0.002	8.1
	Aminoacyl–tRNA hydrolase	J3KQ48	PTRH2	0.0001	5.6
	A–kinase anchor protein 12	Q02952	AKAP12	< 0.0001	Unique
	Fascin	Q16658	FSCN1	< 0.0001	Unique
	ATP–dependent RNA helicase DDX24	Q9GZR7	DDX24	0.0002	Unique
	Aladin	Q9NRG9	AAAS	0.001	Unique
Nitrofurantoin	Probable 28S rRNA (cytosine(4447)–C(5))-methyltransferase	P46087	NOP2	0.002	9.5
	ATPase family AAA domain–containing protein 3A	Q9NVI7	ATAD3A	0.0003	4.5
	Proteasome subunit beta type–3	P49720	PSMB3	0.002	4.8
	H/ACA ribonucleoprotein complex subunit DKC1	O60832	DKC1	0.002	4.9
	40S ribosomal protein S15	K7ELC2	RPS15	0.0004	2.6
	Integrin alpha–6	A0A8C8KBL6	ITGA6	< 0.0001	5.5
	Serine/arginine–rich splicing factor 1	J3KTL2	SRSF1	0.001	3.7
	Caveolae–associated protein 3	E9PIE3	CAVIN3	0.002	6.4
	Membrane–associated progesterone receptor component 1	O00264	PGRMC1	0.001	9.3
	WD40 repeat-containing protein SMU1	Q2TAY7	SMU1	0.0003	16.2
	Oxygen–dependent coproporphyrinogen-III oxidase, mitochondrial	P36551	CPOX	0.004	42.3
	MARCKS–related protein	P49006	MARCKSL1	0.002	11.6
	Aminoacyl–tRNA hydrolase	J3KQ48	PTRH2	0.0001	16.1
	Synaptic vesicle membrane protein VAT–1 homolog	Q99536	VAT1	0.003	6.8
	Sec1 family domain–containing protein 1	A0A7I2V3G4	SCFD1	0.0001	35.0
MIX	Integrator complex subunit 1	Q8N201	INTS1	0.0003	0.2
	Proteasome subunit beta type–3	P49720	PSMB3	0.002	0.4
	Protein disulfide-isomerase TMX3	Q96JJ7	TMX3	0.004	0.4
	Very–long–chain (3R)–3–hydroxyacyl–CoA dehydratase 2	Q6Y1H2	HACD2	< 0.0001	0.5
	Cathepsin B	A0A7P0NGZ6	CTSB	0.002	0.5
	Guanine nucleotide–binding protein G(s) subunit alpha isoforms short	A0A7I2V5R6	GNAS	0.001	0.4
	Albumin	A0A0C4DGB6	ALB	< 0.0001	0.3
	Stomatin–like protein 2, mitochondrial	Q9UJZ1	STOML2	0.001	0.5
	PC4 and SFRS1–interacting protein	O75475	PSIP1	0.001	0.6
	60S acidic ribosomal protein P0	P05388	RPLP0	< 0.0001	0.5
	Sideroflexin–1	Q9H9B4	SFXN1	0.004	0.4
	UDP–glucose:glycoprotein glucosyltransferase 1	Q9NYU2	UGGT1	< 0.0001	2.7
	Protein disulfide–isomerase A4	P13667	PDIA4	< 0.0001	1.9
	Endoplasmin	P14625	HSP90B1	< 0.0001	1.8
	Delta–1–pyrroline–5–carboxylate synthase	P54886	ALDH18A1	< 0.0001	1.7
	Calreticulin	P27797	CALR	< 0.0001	1.6
	Protein disulfide–isomerase A3	A0A8I5KT88	PDIA3	< 0.0001	1.6
	Heterogeneous nuclear ribonucleoprotein R	O43390	HNRNPR	< 0.0001	1.7
	Glutaminase kidney isoform, mitochondrial	O94925	GLS	< 0.0001	1.9
	Histone H1.5	P16401	H1–5	0.004	1.9
	Thioredoxin domain–containing protein 5	Q8NBS9	TXNDC5	< 0.0001	1.8
	Coactosin–like protein	Q14019	COTL1	0.001	2.1
	T–complex protein 1 subunit epsilon	P48643	CCT5	0.001	2.0
	Lysophospholipid acyltransferase 7	Q96N66	MBOAT7	0.001	1.9
	Splicing factor U2AF 65 kDa subunit	P26368	U2AF2	0.002	2.3
	Lamin–B1	P20700	LMNB1	< 0.0001	2.0
	LIM domain and actin–binding protein 1	Q9UHB6	LIMA1	0.0004	2.2
	CD44 antigen	P16070	CD44	< 0.0001	2.1
	Tyrosine–protein phosphatase non–receptor type 1	P18031	PTPN1	0.0003	1.8
	Mitochondrial import receptor subunit TOM70	O94826	TOMM70	0.001	3.2
	Histone H1.4	P10412	H1–4	0.002	3.4
	Thymosin beta–10	P63313	TMSB10	0.003	2.7
	Aminoacyl–tRNA hydrolase	J3KQ48	PTRH2	0.0001	4.9
	Brain acid soluble protein 1	P80723	BASP1	0.001	3.0
	Very–long–chain 3–oxoacyl–CoA reductase	Q53GQ0	HSD17B12	0.0001	3.1
	MARCKS–related protein	P49006	MARCKSL1	0.002	4.6
	ATP–dependent 6–phosphofructokinase, platelet type	Q01813	PFKP	0.001	4.8
	Podocalyxin	O00592	PODXL	< 0.0001	Unique
	Ras–related protein Ral–A	P11233	RALA	< 0.0001	Unique
	F–actin–capping protein subunit beta	B1AK88	CAPZB	< 0.0001	Unique
	CCN family member 1	O00622	CCN1	0.0001	Unique
	ATP–dependent RNA helicase DDX24	Q9GZR7	DDX24	0.0002	Unique
	Purine nucleoside phosphorylase	P00491	PNP	0.001	Unique
	3–ketoacyl–CoA thiolase, peroxisomal	P09110	ACAA1	0.001	Unique

a – protein name in the database (UniProt);

b – accession number in the database (UniProt);

c – denotes gene name.

d – ratio of average signal values of reporter ions treatment / control sample;

Unique – protein identified in the treated samples but not in control

Venn diagrams comparing differentially expressed proteins obtained after treatment with nitrofurantoin (N), hydroquinone (HQ), and their mixture (MIX) after 8 h

Table 6

Complete Gene Ontology (GO) classification of differentially expressed proteins (DEPs) in the cells after treatment with the mixture of nitrofurantoin and the hydroquinone by biological processes

GO biological process	No. of proteins in reference list^*	Genes encoding differentially expressed proteins	P-value	False discovery rate
Glutamine catabolic process (GO:0006543)	3	GLS	1.22×10⁻⁵	3.07×10⁻²
Glutamate biosynthetic process (GO:0006537)	4	GLS	2.43×10⁻⁵	4.60×10⁻²
Cellular biosynthetic process (GO:0044249)	3530	CALR, UGGT1, GLS, ALDH18A1, INTS1, U2AF2, HSP90B1, TXNDC5, PDIA4, PDIA3, STOML2, HSD17B12, CCT5, PSIP1, RPLP0, MBOAT7, HACD2, HNRNPR	2.13×10⁻⁵	9.75×10⁻³
Glutamine family amino acid biosynthetic process (GO:0009084)	15	GLS, ALDH18A1	3.54×10⁻⁶	1.78×10⁻²
Protein folding in endoplasmic reticulum (GO:0034975)	11	CALR, HSP90B1, PDIA3	1.29×10⁻⁶	4.72×10⁻³
Protein folding (GO:0006457)	223	CALR, UGGT1, HSP90B1, TXNDC5, PDIA4, PDIA3, CCT5	3.12×10⁻⁷	1.87×10⁻²
Protein maturation (GO:0051604)	494	CALR, UGGT1, HSP90B1, TXNDC5, PDIA4, PDIA3, STOML2, CCT6	6.00×10⁻⁶	2.27×10⁻²
Response to endoplasmic reticulum stress (GO:0034976)	226	CALR, UGGT1, HSP90B1, PDIA4, PDIA3, PTPN1	6.19× 10⁻⁶	4.59×10⁻²

* Homo sapiens, all genes in database were used as reference list, total No. 20580

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DOI: https://doi.org/10.2478/aiht-2026-77-4146 | Journal eISSN: 1848-6312 (formerly 0004-1254) | Journal ISSN: 0004-1254

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Language: English, Croatian

Page range: 169 - 181

Submitted on: Apr 1, 2026

Accepted on: Jul 1, 2026

Published on: Sep 25, 2026

Published by: Institute for Medical Research and Occupational Health

In partnership with: Paradigm Publishing Services

Keywords:

tandem mass tag labelling

Related subjects:

Medicine,

Basic medical science,

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© 2026 Ana Huđek Turković, Željka Stanečić, Ana Butorac, Vilena Kašuba, Marijana Ćurčić, Antonio Gagić, Ivana Šola, Marija Lovrić, Gordana Rusak, Ksenija Durgo, published by Institute for Medical Research and Occupational Health
This work is licensed under the Creative Commons Attribution 4.0 License.

Volume 77 (2026): Issue 3 (September 2026)

Synergistic effects of combined exposure to hydroquinone and nitrofurantoin in T24 bladder cells: an in vitro toxicology study with implications for risk assessment

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Table 5

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