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

Cell culture and drug treatment

The T24 human urinary bladder carcinoma cell line (ATCC HTB–4), kindly provided by the University of Split Faculty of Chemistry and Technology (Split, Croatia), was selected because of its uroepithelial origin and because both nitrofurantoin and hydroquinone are excreted in urine and are therefore in direct contact with the bladder epithelium. Even though the T24 cell line is neoplastic, it retains key signalling pathways related to oxidative stress, DNA damage response, and apoptosis, and is therefore suitable for mechanistic studies of cytotoxicity, genotoxicity, and proteomic alterations. Furthermore, its genetic and phenotypic stability allows for reproducible assessment of synergistic interactions between compounds.

The cells were cultured in Ham's F-12 with L-glutamine medium supplemented with 10 % foetal bovine serum (FBS) and 1 % antibiotics (penicillin 1000 U/mL, streptomycin 100 mg/mL) and kept at 37 °C in a humidified atmosphere with 5 % CO₂ (Brouwer CH, Luzern, Switzerland). All experiments were conducted on the T24 cells between passages 5 and 15 during their logarithmic growth phase. The cells were treated with nitrofurantoin, hydroquinone, and their mixtures for 8 h (Table 1), which corresponds to the average overnight exposure during sleep. An equal amount of drug-free DMSO was used as negative control, considering that the initial solution of nitrofurantoin was prepared in DMSO.

Chemicals and reagents

Analytical standard of hydroquinone, neutral red dye, 2′,7′–dichlorofluorescein diacetate (DCFH-DA), formic acid, H₂O₂, cytochalasin B, and doxorubicin were purchased from Sigma Aldrich (St. Louis, MO, USA). Ham's F–12 with L-glutamine medium and FBS were obtained from Capricorn Scientific (Ebsdorfergrund, Germany). Nitrofurantoin was procured from Tokyo Chemical Industry Co., Ltd. (Chennai, India). DMSO and ethanol were purchased from Kemika (Zagreb, Croatia). Triethyl ammonium bicarbonate (TEAB) and tandem mass tag (TMT) duplex kit were procured from Thermo Fisher Scientific (Waltham, MA, USA). Acetonitrile was procured from VWR Chemicals (Radnor, PA, USA). Giemsa stain and glacial acetic acid (≥99 %) were obtained from Merck (Darmstadt, Germany).

The concentrations of hydroquinone used in this study are based on concentrations determined in bearberry [Arctostaphylos uva-ursi (L.) Spreng.] extract in our previous study (8). Two concentrations (320 and 900 μg/mL) showed toxic effect and two (20 μg/mL and 50 μg/mL) represent potential therapeutic levels after in vitro digestion of the Uva-ursi extract. Furthermore, we needed to reduce the therapeutic concentrations for the cytokinesis-block micronucleus (CBMN) cytome assay to 5 and 10 μg/mL, as explained below.

The nitrofurantoin concentrations were selected based on the recommended dosing regimens (8), with 330 and 670 μg/mL corresponding to the lowest and highest therapeutic doses, respectively, whereas the 167 μg/mL concentration corresponds to the prophylactic oral dose of 100 mg.

Neutral red cytotoxicity assay

Cells were seeded in 96-well plates at a concentration of 10⁵ cells/mL and grown for 24 h before the 8-hour treatment. After the treatment, they were washed twice with phosphate-buffered saline (PBS). The cytotoxic effect was determined using the neutral red assay following the protocol described by Babich and Borenfreund (9). Absorbance was measured spectrophotometrically at 540 nm using a Fluostar OPTIMA plate reader (BMG Labtech, Durham, NC, USA). We conducted the experiment using two different approaches: one without recovery, where cell survival was measured immediately after treatment, and the other with recovery, where the medium was added and cell survival measured 24 h after the treatment. The percentage of surviving cells was calculated in comparison to the negative control (100 %).

Reactive oxygen species determination

Cells were seeded as described above and the DCFH-DA fluorometric assay used to measure the amount of reactive oxygen species (ROS) with and without 24-hour recovery after treatment as described by Silveira et al. (10). Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The results are expressed as increase or decrease in fluorescence intensity relative to negative control. Cells treated with 10 % H₂O₂ for 15 min served as positive control.

Cytokinesis-block micronucleus cytome (CBMN Cyt) assay

T24 cells (1×10⁴ cells/mL) were seeded in 24-well plastic plates with 14 mm diameter oval glass coverslips (Paul Marienfeld GmbH, Lauda-Königshofen, Germany) containing complete medium described above. Cells were treated for two and eight hours with nitrofurantoin (167 μg/mL), hydroquinone (5 or 10 μg/mL), or one of their combinations. For positive control we used doxorubicin (0.4 μg/mL), while the negative control was treated with 0.16 % v/v DMSO. The CBMN cytome assay was performed as described by Savio et al. (11), following the OECD Guideline No. 487 (12). Cytochalasin B was used at 3 μg/mL for 24 h. After processing cell cultures, prepared slides were dried and refrigerated at 20 °C until microscopy in oil immersion (1000×magnification; Olympus CX41, Tokyo, Japan). The frequencies of micronuclei (MN), nuclear buds (NB), and nucleoplasmic bridges (NPB) were measured in six thousand binucleated cells as described by Fenech and Crott (13), while the mitotic index and apoptotic and necrotic rates were measured as described by Eastmond and Tucker (14) in thousand cells per each experimental point.

Protein analysis

The cells were seeded in 6-cm cell culture plates at a density of 1×10⁶ cells per plate and grown for 24 h under conditions described above. After incubation, the cells were treated with nitrofurantoin (167 μg/mL) or hydroquinone (20 μg/mL) or their mixture for 8 h and then rinsed with PBS, resuspended in lysis buffer (1 % sodium dodecyl sulphate in 100 mmol/L TEAB), and sonicated on ice using a Sonopuls HD 3200 homogeniser (Bandelin, Berlin, Germany) in three cycles at 10 s amplitudes of 30 %, separated by 20 s cooling pauses, as described in the user manual (15).

The obtained suspension was then centrifuged at 10000×g and 4 °C for 10 min and the supernatant analysed for total protein concentration using the Bradford assay (16).

Reduction, alkylation, digestion, and TMT labelling followed the manufacturer's instructions (15). Three biological replicas were analysed per group.

Peptide separation and mass spectrometry were performed using the SCIEX TripleTOF 6600+ system equipped with an Optiflow Turbo V Ion Source (Sciex, Framingham, MA, USA). Chromatographic separation was performed on a Phenomenex (Torrance, CA, USA) Luna Omega Polar C18 column (100 A, 150×0.3 mm) at 27 °C. The flow rate was set to 5 μL/min. Mobile phases A and B consisted of 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile, respectively. Gradient elution was set as follows: 5–30 % B (0–68 min), 30–40 % B (68–73 min), 80 % B (73–78 min), 80–3 % B (78–79 min), and 3 % B (79–87 min). The ionisation source operated in positive ion mode with the following parameters: ion spray voltage of 4500 V, nebuliser gas 1 at 30 psi, nebuliser gas 2 at 35 psi, and curtain gas at 30 psi. The mass spectrometer scanned full spectra (m/z 400–1500) for 250 ms. Top 50 precursors were selected for MS/MS fragmentation (m/z 100–2000) with a charge state between +2 and +5 and counts above a minimum threshold (100 counts per second). Rolling collision was employed with the energy spread of 5. After MS acquisition, the obtained MS/MS spectra were used for database search using the ProteinPilot Software 5.0.2 (AB Sciex, Marlborough, MA, USA). The Homo sapiens proteome database was downloaded from the UniProt database (entry number 26610; revised SwissProt sequences; accessed on 1 August 2024). Search parameters included one missed trypsin cleavage, carbamidomethylation, and TMT6plex as fixed modifications. Precursor ion and fragment ion mass tolerance were set to 0.05 and 0.10 Da, respectively. The results were refined to include only proteins meeting the criterion of having a minimum of two identified peptide sequences and a false discovery rate of less than 1 %.

Enrichment analysis was performed on sets of genes that were up-regulated or down-regulated under the studied conditions using the Gene Ontology (GO) PANTHER 18.0 engine (University of Southern California, Los Angeles, CA, USA) which employs Fisher's exact test and false discovery rate correction. Overlapping proteins between the conditions were illustrated with the Venn diagram (17).

Statistical analysis

Dose-response [benchmark dose (BMD) modelling] of the single hydroquinone (0–900 μg/mL) and single nitrofurantoin concentrations (0–670 μg/mL) and their mixtures was analysed on the PROAST software package (RIVM, Bilthoven, The Netherlands) as described by More et al. (18).

The effect of mixtures in relation to individual compounds was analysed using the factorial regression model within the STATISTICA software package version 7.0 (StatSoft, Tulsa, OK, USA) according to the following equation:

To evaluate treatment differences in neutral red and DCHF-DA assays, we ran one-way ANOVA and post-hoc Tukey's HSD test. The Kolmogorov-Smirnov test was used to evaluate the distribution of data for normality and the chi-squared test to compare the results of the CBMN cytome test.

Statistical analysis of proteomics data was performed using the Scaffold Q+S program 5.3.0 (Proteome Software, Portland, OR, USA). Quantification was based on the reporter ion signal employing ratio-based normalisation and Permutation tests.

Statistical significance for all tests was set to P<0.05.

Cytotoxicity and ROS findings

Compared to control, the highest therapeutic dose of nitrofurantoin (670 μg/mL) significantly reduced T24 cell survival to below 80 % after 8 h of treatment (P<0.05; Figure 1a). Similar effects were observed after the 24-hour recovery period, suggesting that the damage at the cellular level is permanent. Both therapeutic doses exhibited lower antioxidative activity in unrecovered T24 cells, possibly contributing to their lower survival.

Figure 1

DMSO reduced T24 cell survival by 1.5 %, and all DMSO concentrations in nitrofurantoin samples (0.16–0.6 %) were far below toxic concentrations.

Generally, our results agree with previous observations by Vumma et al. (19), who found that nitrofurantoin did not affect the viability of 5637 (HTB-9) urinary bladder cells at concentrations of 30 and 300 μg/mL after 2, 4, and 12-hour exposure. Although both 5637 and T24 are bladder carcinoma cell lines, they primarily differ in their aggressiveness and genetic profiles. 5637 cells are derived from a less aggressive, non-invasive carcinoma, while T24 cells originate from a more aggressive, invasive carcinoma with a mutation in the H-Ras gene (20). Conversely, nitrofurantoin was reported to inhibit the growth of FANFT-induced murine bladder tumour (MBT2) and a human transitional cell carcinoma cell line (GIBB) in the concentration range of 30–120 μg/mL during an in vitro treatment lasting 2–9 days (21). T24 and MBT2 cells may yield different results due to species-specific differences in genetics, tumour induction, immune system interactions, and metabolic processes, with T24 being human-derived and MBT2 being murine. Also, the difference in results may be due to 6–25 times longer exposure times than those used in our study.

The two highest concentrations of hydroquinone tested in our study correspond to the highest bladder tissue exposure scenario from recommended treatment doses. Both significantly reduced survival and affected the morphology of T24 cells. The toxic effect was enhanced during cell recovery, which suggests that hydroquinone enters cells and initiates processes that continue during cell reproduction. Lee et al. (22) reported that 44 μg/mL of free hydroquinone induced acute toxicity within 4 h in human kidney proximal tubule epithelial cells (HK-2) and the transformed human liver epithelial (THLE-2) cell line. In contrast, our study revealed that concentrations of 20 and 50 μg/mL reduced survival only after 8 h in T24 cells, indicating higher resistance compared to normal cells and suggesting reversible damage from short (<8 h) exposures.

We found that hydroquinone started to exert cytotoxic and oxidative effects at ≥50 μg/mL in unrecovered T24 cells. Following the 24-hour cell recovery, the cytotoxic effect remained visible (Figure 1b), but the redox status recuperated and did not differ from the negative control. However, the highest concentrations of hydroquinone (corresponding to consuming one cup of bearberry leaf tea or a daily consumption (320 or 900 μg/mL, respectively) exhibited cytotoxic and oxidative effects regardless of cell recovery. It is possible that hydroquinone-induced disruption of T24 cell membrane integrity caused irreparable damage unsuitable for ROS measurement. These concentrations caused permanent cell damage, as T24 cell survival decreased to ~30 %, regardless of exposure time. However, at lower concentrations over 8 h, reversible damage occurred with no lasting effects after recovery.

The combined effects of nitrofurantoin and hydroquinone were significant in unrecovered T24 cells at all concentrations, affecting both the survivability and oxidative status (Figure 1, panel c1), while in the recovered cells, these effects subsided only at the lowest hydroquinone concentrations (Figure 1, panel c2). Table 2 shows a synergistic effect of the combination on survival in recovered cells and on oxidative status in unrecovered cells. However, the very low β₁₂ factor values raise doubt if these compounds interacted synergistically in all mixture combinations with different concentrations and ratios.

Genome instability effects

The CBMN Cyt assay used in this study to elucidate the toxic effects of hydroquinone and nitrofurantoin provides an integrated assessment of multiple cellular endpoints, including DNA and chromosomal damage, as well as disturbances in intracellular proteins involved in mitotic spindle assembly and cytoskeletal organisation (23). Previous studies have suggested that hydroquinone may disrupt intracellular protein components, thereby contributing to cellular dysfunction. Aspengren et al. (24) have shown that hydroquinone (1.1–5.5 μg/mL) disrupts the dermal melanophores of Xenopus laevis by reorganising microtubules. Oxidative stress alters the cytoskeleton, and maintaining this balance is crucial for normal cellular function. In a study by Kim et al. (25), the high hydroquinone concentration of 50 μmol/L (corresponding to 5.51 μg/mL) increased apoptosis of human lymphoblastic leukaemia cells (Jurkat cells) via ERK (contributes to cell growth while inhibiting apoptosis) and caspase inhibition, while the lower concentration of 5 μmol/L (0.55 μg/mL) increased the proportion of cells in the S phase of the cell cycle, possibly due to effects on microtubule distribution.

We found that hydroquinone (20 and 50 μg/mL) negatively affected the adhesion of T24 cells on glass slides 2 h after treatment, which made it impossible to analyse them. Moreover, nitrofurantoin slowed down T24 cell division, resulting in insufficient binucleated cells in the treated samples. Consequently, a follow-up experiment was carried out for both 2 and 8 h using lower doses of hydroquinone alone (5 and 10 μg/mL) as well as a combination of hydroquinone and nitrofurantoin for 2 h (5 or 10 μg/mL combined with 167 μg/mL).

The results of the CBMN Cyt assay with the lowered hydroquinone concentrations (5 and 10 μg/mL) are presented in Table 3. The MN count was significantly higher in all treated samples at both exposure times compared to the negative control. The highest count was observed in the sample treated with the mixture of hydroquinone (5 μg/mL) and nitrofurantoin (167 μg/mL) for 2 h (MIX 1) and was significantly higher than the sum of MNs in samples treated with individual compounds (P=0.001; chi-squared test), which we consider a synergism because the effect of the mixture is greater than the sum of individual compounds.

In the sample treated with the mixture of 10 μg/mL hydroquinone with nitrofurantoin (MIX 2) for 2 h, hydroquinone had a significant effect on the adhesion of T24 cells, rendering cell scoring impossible (Table 3). Furthermore, due to the insufficient number of binucleated cells, the results for hydroquinone (10 μg/mL) are expressed for total of 3000 (instead of 6000) binucleated T24 cells.

Exposure duration did not affect the MN counts in the samples treated with hydroquinone (Table 3).

The occurrence of NBs in untreated control binucleated T24 cells was quite low (1.3±0.75 per 1000 cells). Treating T24 bladder cancer cells with nitrofurantoin and hydroquinone, as well as their combination for 2 h led to a significant increase in NBs compared to untreated control. The highest NB frequency was observed in the sample treated with MIX 1, which is significantly higher than in samples treated with individual compounds (P=0.036; chi-squared test), indicating a synergistic effect. NB frequency in other samples ranged from 5.0±1.55 to 8.5±2.2 per 1000 binucleated cells (Table 3).

Jurica et al. (26) observed no impact on the formation of micronuclei in lymphocytes after a 24-hour treatment with the 8 μg/mL concentration of hydroquinone. However, lymphocytes had statistically significantly more NBs compared to the negative control, while the concentrations of 140 and 280 μg/mL completely blocked lymphocyte division due to nuclear budding occurring during the S-phase of the cell cycle. It is believed that DNA repair results in excessive formation of nuclear buds, concentrated in the peripheral part of the nucleus (27). The differences between from our findings may therefore be due to differences in cell lines used for research.

NPB frequency in all treated cells was significantly higher than in untreated control, and the highest was recorded for 10 μg/mL hydroquinone alone (Table 3). It was also significantly higher than in T24 cells treated with 5 μg/mL hydroquinone alone, which was not the case with MN and NB.

MIX 1 yielded lower NPB frequency than 10 μg/mL hydroquinone alone, but the effect was significantly higher than with nitrofurantoin alone. Results obtained for MIX 1 suggest that nitrofurantoin and hydroquinone in the mixture act synergistically (P=0.022, chi-squared test).

It was not possible to determine the number of NB and NPB in the sample treated with a mixture of nitrofurantoin (167 μg/mL) and hydroquinone (10 μg/mL; MIX 2) for 2 h due to an insufficient number of binucleated cells on the slide. The 10 μg/mL hydroquinone significantly affected the adhesion of T24 cells, so the results are expressed per 500 binucleated cells instead of 3000. The number of NB is statistically significantly higher in the sample treated with a concentration of hydroquinone of 10 μg/mL for 8 h compared to the lower concentration, while there is no statistically significant difference in the number of NPB.

Exposure duration (2 and 8 h) did not significantly affect the number of NB and NPB in the samples treated with hydroquinone.

Table 4 shows significantly lower nuclear division index (NDI) in the cells exposed for 2 h to both nitrofurantoin concentrations and to both mixtures. Furthermore, mixture NDI was significantly lower than with either compound alone. Considering that hydroquinone treatment did not significantly lower the NDI, our results suggest that it potentiates the negative effect of nitrofurantoin in the mixture.

The 8 h exposure to nitrofurantoin and hydroquinone had an even stronger effect. All the above also applies to the value of the replication index (RI, Table 4). Compared to negative control, the only significant rise in apoptotic T24 cells was determined in the group treated with 10 μg/mL hydroquinone for 8 h (25/3000) (P<0.05, chi-squared test).

The cytostatic effects, cytokinesis-block proliferation index (CBPI) in particular, completely reflect NDI findings and are dose-dependent. Significantly higher percentage of cytostasis caused by both mixtures in T24 cells after 2 h of exposure compared to nitrofurantoin alone suggests that hydroquinone in the mixture potentiates the cytostatic activity of nitrofurantoin.

Cell kinetics findings suggest that nitrofurantoin inhibits or halts the growth and division of T24 cells. This effect is dose-dependent and increases with longer treatment time. One study (28) confirmed the genotoxic potential of nitrofurantoin in young and adult mice in vivo, showing that young mice (3 weeks old) were more sensitive to its genotoxic effects than adult mice (8 weeks old) and that response in young mice lasted longer. The application of the CBMN test in vivo revealed a statistically significantly higher number of micronuclei in reticulocytes from mice treated with 5, 10, and 50 mg/kg nitrofurantoin compared to the control group.

Additionally, previous study (26) demonstrated a significant disruption in lymphocyte kinetics by hydroquinone at a concentration of 8 μg/mL, which supports this study's findings regarding its impact on chromosomal aberrations and abnormal mitotic processes, including MN formation and sister chromatid alterations without direct evidence of mutagenicity.

Protein analysis

Table 5 shows proteins whose expression changed significantly in T24 cells treated for 8 h v untreated cells, as identified with the permutation test. In total, 15 differentially expressed proteins (DEPs) were identified after nitrofurantoin treatment, 28 after hydroquinone treatment, and 44 after treatment with their mixture (167 μg/mL nitrofurantoin and 20 μg/mL hydroquinone).

The Venn diagram (Figure 2) shows that only two DEPs overlap between all conditions (MARCKSL1 and PTRH2), 12 between the mixture and hydroquinone (CTSB, GNAS, ALB, SFXN1, UGGT1, ALDH18A1, MBOAT7, CD44, PTPN1, H1-4, PFKP, and DDX24), one between nitrofurantoin and the mixture (PSMB3), and one between nitrofurantoin and hydroquinone (VAT1). Furthermore, 29 DEPs belong exclusively to the MIX group (INTS1, TMX3, HACD2, STOML2, PSIP1, RPLP0, PDIA4, HSP90B1, CALR, PDIA3, HNRNPR, GLS, H1-5, TXNDC5, COTL1, CCT5, U2AF2, LMNB1, LIMA1, TOMM70, TMSB10, BASP1, HSD17B12, PODXL, RALA, CAPZB, CCN1, PNP, and ACAA1), 11 to the nitrofurantoin alone group (NOP2, ATAD3A, DKC1, RPS15, ITGA6, SRSF1, CAVIN3, PGRMC1, SMU1, CPOX, and SCFD1), and 13 to the hydroquinone alone group (ALYREF, TPM3, SLC39A3, HNRNPU, SUCLG1, HMGN2, PLOD2, FLNC, MANF, LMO7, AKAP12, FSCN1, and AAAS).

Figure 2

The complete Gene Ontology (GO) database classification of DEPs (Table 6) shows that the mixture significantly affected the expression of proteins related to glutamine catabolism (GO:0006543), glutamate biosynthesis (GO:0006537), glutamine family amino acid biosynthesis (GO:0009084), cellular biosynthesis (GO:0044249), protein folding in endoplasmic reticulum (GO:0034975), protein folding (GO:0006457), protein maturation (GO:0051604), and response to endoplasmic reticulum stress (GO:0034976).

Our proteomic analysis reveals the up-regulation of MARCKS-related protein and peptidyl-tRNA hydrolase 2 over 8 h. MARCKS-related protein controls cell movement by regulating actin cytoskeleton homeostasis and the formation of filopodia and lamellipodia. In its unphosphorylated form, it induces cell migration. When phosphorylated, it facilitates the formation and stabilisation of actin bundles, which, in turn, reduces actin plasticity and limits cell movement (29). Mitochondrial peptidyl-tRNA hydrolase 2 regulates the function of two transcriptional regulators, AES and TLE1, and through them promotes caspase-independent apoptosis (29).

In our previous study (8), we investigated proteome changes in human bladder T24 cells induced by hydroquinone after 2 h, and the results indicated increased expression of proteins related to the glycolysis (GO:0006096), response to stimulus (GO:0050896), and translational initiation (GO:0006413). In this study, we prolonged the treatment to 8 h and detected a completely changed, differentially expressed set of proteins (DEPs). Although gene ontology analysis did not classify the DEPs into specific biological processes, according to the 2023 Universal Protein Knowledgebase (28), fascin, filamin-C, and tropomyosin alpha-3 chain are involved in the actin-binding molecular function (GO:0003779). Fascin is involved in the organisation of actin filament bundles and the formation of microspikes, membrane ruffles, and stress fibres. It plays an important role in forming a diverse set of cell protrusions, such as filopodia, and in cell motility and migration. Fascin also mediates the reorganisation of the actin cytoskeleton. Filamin-C plays a central role in muscle cells, likely as a large actin-cross-linking protein. It may be involved in reorganising the actin cytoskeleton in response to signalling events and is essential for normal myogenesis and for maintaining the structural integrity of muscle fibres. Tropomyosin alpha-3 chain is an actin-binding protein implicated in stabilising cytoskeletal actin filaments. At the molecular level, hydroquinone caused changes in the T24 cytoskeleton, quite likely because of the activated cytotoxic mechanisms.

After nitrofurantoin treatment, gene ontology analysis did not classify the set of DEPs into specific biological processes. However, our results suggest that nitrofurantoin primarily affected proteins involved in RNA metabolism (GO:0016070) such as ribosomal protein S15, H/ACA ribonucleoprotein complex subunit DKC1, 28S rRNA (cytosine(4447)-C(5))-methyltransferase, serine/arginine-rich splicing factor 1, WD40 repeat–containing protein SMU1, and proteasome subunit beta type-3. Probable 28S rRNA (cytosine(4447)-C(5))-methyltransferase may play a role in regulating the cell cycle and the increased nucleolar activity associated with cell proliferation (29). H/ACA ribonucleoprotein complex subunit DKC1 is a protein that catalyses the pseudouridylation of rRNA but can also promote cell-to-cell and cell-to-substratum adhesion, increase cell proliferation, and lead to cytokeratin hyper-expression (29). Proteasome subunit beta type-3 plays a key role in maintaining protein homeostasis by removing misfolded or damaged protein, and WD40 repeat-containing protein SMU1 is essential for normal mitotic spindle assembly and mitosis (29).

Synergistic activity of nitrofurantoin and hydroquinone in mixture

Our study shows that the combination of nitrofurantoin (167 μg/mL) and hydroquinone (5 μg/mL) had a synergistic genotoxic effect in T24 cells compared to the individual compounds. Moreover, hydroquinone enhanced the cytostatic action of nitrofurantoin, particularly when the exposure was prolonged to 8 h. This finding is relevant, as it indicates a potential interaction that may alter the antibiotic's effectiveness. In contrast, a previous study examined the antioxidant properties of Rosmarinus officinalis, Cynara scolymus L., and Hedera helix extracts during nitrofurantoin therapy, and reported that these plant extracts reduced the prooxidative impact (~50 %) of nitrofurantoin (30).

The up-regulation of proteins involved in biological processes such as the glutamine catabolism (GO:0006543), glutamate biosynthesis (GO:0006537), cellular biosynthesis (GO:0044249), and glutamine family amino acid biosynthesis (GO:0009084) strongly suggests that the synergistic activity of nitrofurantoin and hydroquinone activates this mechanism to prevent severe damage to biomolecules (31). The synergistic activity of the combination also activated biological processes such as protein folding in the endoplasmic reticulum (GO:0034975), protein folding (GO:0006457), protein maturation (GO:0051604), and response to endoplasmic reticulum stress (GO:0034976). The endoplasmic reticulum plays a crucial role in protein quality control and homeostasis, including degradation, protein chaperones, and autophagy. The accumulation of misfolded and unfolded proteins triggers stress in endoplasmic reticulum to restore proteostasis (32).