Role of oxidative stress in the chemical structure-related genotoxicity of nitrofurantoin in Nrf2-deficient gpt delta mice.

Despite its antimicrobial activity, nitrofurantoin (NFT) is a renal carcinogen in rats. Oxidative stress induced by reduction of the nitro group of NFT may contribute to its genotoxicity. This is supported by our recent results indicating that the structure of the nitrofuran plays a key role in NFT-induced genotoxicity, and oxidative DNA damage is involved in renal carcinogenesis. Nuclear factor erythroid 2-related factor 2 (NRF2) regulates cellular responses to oxidative stress. To clarify the role of oxidative stress in the chemical structure-related genotoxic mechanism of NFT, we performed reporter gene mutation assays for NFT and 5-nitro-2-furaldehyde (NFA) using Nrf2-proficient and Nrf2-deficient gpt delta mice. NFT administration for 13 weeks resulted in a significant increase in 8-hydroxydeoxyguanosine (8-OHdG; a marker of oxidative stress) and gpt mutant frequency only in the kidneys of Nrf2-/- mice. The mutation spectrum, characterized by increased substitutions at guanine bases, suggested that oxidative stress is involved in NFT-induced genotoxicity. However, NFA did not increase the mutation frequency in the kidneys, despite the increased 8-OHdG in NFA-treated Nrf2-/- mice. Thus, it is unlikely that oxidative stress is involved in the genotoxic mechanism of NFA. These results imply that nitro reduction plays a key role in the genotoxicity of NFT, but the lack of a role of oxidative stress in the genotoxicity of NFA indicates a potential role of side chain interactions in oxidative stress caused by nitro reduction. These findings provide a basis for the development of safe nitrofurans.

Introduction

Nitrofurans are antimicrobial compounds that contain a nitro group at the 5-position of the furan ring and an amine or hydrazide side chain derivative (Fig. 1). Some nitrofurans are prohibited from use in veterinary medicine in Japan owing to their genotoxic and carcinogenic potential[1], [2], [3], [4]. However, new nitrofurans with various hydrazide derivatives on the side chain are being developed, given their easy synthesis and high antimicrobial activity[5], [6]. Therefore, it is necessary to clarify the chemical structure-related genotoxicity of nitrofurans to facilitate risk assessments for human applications.

Fig. 1.

Chemical structures of NFT and NFA.

One nitrofuran group, nitrofurantoin (NFT), is synthesized by the condensation of 5-nitro-2-furaldehyde (NFA) (Fig. 1) and 1-aminohydantoin and is a renal carcinogen in rats[7]. The formation of reactive oxygen species (ROS) or intermediates resulting from the reduction of the nitro group of NFT is thought to exert antibacterial activity[8], [9], [10]. Accordingly, we hypothesized that oxidative stress is involved in NFT-induced renal carcinogenesis. We recently demonstrated significant increases in the levels of 8-hydroxydeoxyguanosine (8-OHdG), an oxidized DNA lesion, and gpt mutant frequencies (MFs) with substitutions at guanine bases in the kidneys of gpt delta rats treated with NFT[11]. However, the 1-aminohydantoin side chain did not increase 8-OHdG levels or gpt MFs[11]. NFA containing a nitro group, similar to NFT, did not increase 8-OHdG levels but increased gpt MFs in the kidneys of gpt delta rats with different mutation spectra from those for NFT[11]. Accordingly, the relationship between NFT-induced oxidative stress and its chemical structure remains unclear[11].

The redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) regulates cellular responses to oxidative stress. NRF2 is anchored in the cytoplasm by Kelch-like ECH-associated protein 1 (KEAP1), which also mediates the proteasomal degradation of NRF2. Oxidative stress causes the dissociation of NRF2 from KEAP1 and leads to NRF2 translocation into the nucleus, where it can bind to the antioxidant response element (ARE) and consequently transactivate ARE-bearing genes encoding antioxidant-related enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1), and glutathione S-transferase[12], [13]. Thus, the NRF2-ARE pathway has broad protective effects against oxidative stress. Nrf2-deficient mice clearly show greater sensitivity to various toxicants, as evidenced by induction of the oxidative stress response following exposure to acetaminophen, 4-vinylcyclohexene diepoxide, pentachlorophenol, 2-amino-3-methylimidazo[4,5-f]quinoline, ferric nitrilotriacetate, and piperonylbutoxide[14], [15], [16], [17], [18], [19], [20].

In the present study, the role of oxidative stress in the chemical structure-related genotoxicity of NFT was determined using Nrf2-proficient and Nrf2-deficient mice exposed to NFT or NFA for 13 weeks, followed by reporter gene mutation assays[21], [22] and measurements of 8-OHdG levels in the kidney.

Materials and Methods

Chemicals

NFT (C8H6N4O5, MW 238.2, CAS No. 67-20-9) and NFA (C5H3NO4, MW 141.08, CAS No. 698-63-5) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA) and were suspended in 0.5 w/v% methyl cellulose 400 cP solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Suspensions of the test chemicals were used at a volume of 10 mL/kg body weight (BW), based on BW on the day of chemical administration to Nrf2+/+ or Nrf2−/−gpt delta mice.

Animals, diet, and housing conditions

The study protocol was approved by the Animal Care and Utilization Committee of the National Institute of Health Sciences. Nrf2-deficient mice with the C57BL/6J background established by Itoh et al.[23] were crossed with gpt delta mice with the C57BL/6J background (Japan SLC, Shizuoka, Japan). Nrf2−/−gpt delta mice and Nrf2+/+gpt delta mice were then obtained from the F1 generation and genotyped by polymerase chain reaction (PCR) with DNA collected from the tail of each mouse. All mice were housed in polycarbonate cages (5 mice per cage) with hard wood chips for bedding in a conventional animal facility maintained at a controlled temperature (23 ± 2°C) and humidity (55 ± 5%), with 12 air changes per hour and a 12-h light/dark cycle. Mice were given free access to a basal diet (CRF-1, Charles River Laboratories Japan, Kanagawa, Japan) and tap water.

Experimental design

Eight-week-old male mice of each genotype were divided into five groups (four or five mice per group), i.e., two groups each administered NFT or NFA by gavage for five consecutive days and a control group administered vehicle alone, and the total administration period was 13 weeks. For daily doses, 70 and 35 mg/kg NFT were used. The maximum tolerated dose of NFT was 70 mg/kg in a preliminary dose selection study. No remarkable changes were observed in the general condition of mice treated with NFT at a dose of 70 mg/kg in the preliminary study. The daily doses of NFA were set to 41 and 21 mg/kg, the same molar doses used for NFT. BW was measured every week. At the end of administration for 13 weeks, animals were euthanized by exsanguination under isoflurane (Mylan Inc., Tokyo, Japan) anesthesia, and the bilateral kidneys were collected and weighed. A portion of the kidney tissues was frozen with liquid nitrogen and stored at −80°C for an in vivo mutation assay, 8-OHdG measurements, and western blotting. Another portion of the collected kidney tissues was homogenized in ISOGEN (Nippon Gene, Tokyo, Japan) and stored at −80°C until use for the isolation of total RNA.

in vivo mutation assays

6-Thioguanine (6-TG) and Spi– selection were performed using the methods described by Nohmi, et al[21]. Briefly, genomic DNA was extracted from the kidneys of animals in each group using a RecoverEase DNA Isolation Kit (Agilent Technologies, Santa Clara, CA, USA), and lambda EG10 DNA (48 kb) was rescued as phages by in vitro packaging using Transpack Packaging Extract (Agilent Technologies). For 6-TG selection, packaged phages were incubated with Escherichia coli YG6020, which expresses Cre recombinase, and converted to plasmids carrying gpt and chloramphenicol acetyltransferase genes. Infected cells were mixed with molten soft agar and poured onto agar plates containing chloramphenicol and 6-TG. To determine the total number of rescued plasmids, infected cells were also poured on plates containing chloramphenicol without 6-TG. The plates were then incubated at 37°C for selection of 6-TG-resistant colonies, and the gpt MF was calculated by dividing the number of gpt mutants after clonal correction by the number of rescued phages. The gpt mutations were characterized by the amplification of a 739-bp DNA fragment containing the 456-bp coding region of the gpt gene[21] and sequencing the PCR products using an Applied Biosystems 3730xl DNA Analyzer (Life Technologies Corporation, Carlsbad, CA, USA). For Spi– selection, packaged phages were incubated with E. coli XL-1 Blue MRA for survival titration and E. coli XL-1 Blue MRA P2 for mutant selection. Infected cells were mixed with molten lambda-trypticase agar plates. The next day, plaques (Spi– candidates) were punched out with sterilized glass pipettes, and the agar plugs were suspended in SM buffer. The Spi– phenotype was confirmed by spotting the suspensions on three types of plates where the XL-1 Blue MRA, XL-1 Blue MRA P2, or WL95 P2 strain was spread on soft agar. Spi– mutants forming clear plaques were counted on every plate.

Measurement of 8-OHdG

Renal DNA of Nrf2−/−gpt delta mice and Nrf2+/+gpt delta mice was extracted and digested as described previously[24]. Briefly, nuclear DNA was extracted using a DNA Extractor WB Kit (Wako Pure Chemical Industries). To prevent artefactual oxidation in the cell lysis step, deferoxamine mesylate (Sigma-Aldrich) was added to the lysis buffer. DNA was digested to deoxynucleotides by treatment with nuclease P1 and alkaline phosphatase using an 8-OHdG Assay Preparation Reagent Kit (Wako Pure Chemical Industries). The levels of 8-OHdG (8-OHdG/105dG) were measured for three randomly selected mice in each group by high-performance liquid chromatography using an electrochemical detection system (Coulochem II, ESA, Bedford, MA, USA) as previously reported[25]. Because of the quite small amount of kidney samples applied for measurement, the data were obtained from only one mouse in the 41 mg/kg NFA group.

RNA isolation and quantitative real-time PCR for mRNA expression

Total RNA was extracted using ISOGEN according to the manufacturer’s instructions. cDNA copies of total RNA were obtained using a High-Capacity cDNA Reverse Transcription Kit (Life Technologies).

All PCRs were performed using an Applied Biosystems 7900HT FAST Real-Time PCR System with primers for mouse Nqo1 obtained from TaqMan® Gene Expression Assays and TaqMan® Rodent GAPDH Control Reagents. Expression levels were calculated by the relative standard curve method and were determined relative to Gapdh levels. Data are presented as fold-change values of treated samples relative to controls.

Protein extraction, SDS-PAGE, and western blotting

The kidneys from all animals were homogenized using a Teflon homogenizer with ice-cold RIPA lysis buffer (Wako Pure Chemical Industries) containing mammalian protease inhibitor cocktail. Samples were homogenized and centrifuged at 15,000 × g for 30 min, and the resulting supernatants were used. Protein concentrations were determined using an Advanced Protein Assay (Cytoskeleton, Denver, CO, USA) with bovine serum albumin as a standard. Samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.45-μm PVDF membranes (Millipore, Billerica, MA, USA). For the detection of target proteins, membranes were incubated with an anti-NQO1 polyclonal antibody (1:1,000; Abcam, Cambridge, UK) and anti-β-actin monoclonal antibody (1:3,000; Abcam) at 4°C overnight. Secondary antibody incubation was performed using horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody at room temperature. Protein detection was facilitated by chemiluminescence using ECL Plus (GE Healthcare Japan Ltd., Tokyo, Japan).

Statistical analysis

Data are presented as the mean ± standard deviation (SD). Statistical analyses of differences in BWs, kidney weights, 8-OHdG levels, mRNA expression levels, gpt and Spi– MFs, and gpt-mutation spectra relative to the values of the control group of mice of the same genotype were analyzed by Dunnett’s multiple comparison test. Comparison between mRNA expression levels of each control group of Nrf2-proficient and Nrf2-deficient mice were made using Student’s t-test. P<0.05 was considered significant.

Results

Body and kidney weights

Body and kidney weights of Nrf2-proficient and Nrf2-deficient mice treated with NFT or NFA for 13 weeks are summarized in Fig. 2 and Table 1. For both genotypes, there were no significant differences in body and kidney weights between treated and untreated mice.

Fig. 2.

Growth curves for Nrf2+/+ (left panel) and Nrf2−/− (right panel) mice treated with NFT or NFA for 13 weeks. For both genotypes, there were no significant differences in body weight between treated and untreated mice.

Table 1.

Final Body and Kidney Weights of Male Nrf2+/+ or Nrf2−/−gpt Delta Mice Treated with NFT or NFA for 13 Weeks

Quantitative real-time PCR and western blotting analyses of Nqo1

For both genotypes, the mRNA expression level of Nqo1 was not significantly influenced by NFT or NFA treatment. In Nrf2-deficient mice, however, the Nqo1 mRNA expression level was significantly lower than that in Nrf2-proficient mice (Fig. 3).

Fig. 3.

Changes in the Nrf2-target gene Nqo1 at the mRNA (A) and protein levels (B). (A) Data are presented as means ± SD. †mRNA expression levels in the Nrf2−/− control group were significantly different (P<0.05) from levels in the Nrf2+/+ control group by Student’s t-test.

Furthermore, at the protein expression level, NQO1 was not affected by NFT or NFA treatment. In Nrf2-deficient mice, however, the NQO1 protein expression level was lower than that in Nrf2-proficient mice (Fig. 3).

8-OHdG levels in kidney DNA

8-OHdG levels in Nrf2-deficient mice treated with 70 mg/kg NFT were significantly higher than those in control mice. 8-OHdG levels in Nrf2-deficient mice treated with NFA showed tendencies toward increasing in a dose-dependent manner, although they were not statistically significant because of insufficiency of samples in the 41 mg/kg NFA group. No increase was observed in Nrf2-proficient mice treated with NFT or NFA at any dose (Fig. 4).

Fig. 4.

8-OHdG levels in the kidneys of Nrf2+/+ or Nrf2−/−gpt delta mice treated with NFT or NFA for 13 weeks. Data are presented as means ± SD for 3 mice in the groups treated with other than 41 mg/kg NFA. In the 41 mg/kg NFA group, the data obtained from one mouse are presented. *Significantly different (P<0.05) from levels in the relative control group by Dunnett’s test.

in vivo mutation assay

Results of the gpt assay for the kidneys of Nrf2-proficient and Nrf2-deficient mice treated with NFT or NFA are shown in Tables 2, 3, 4. The gpt MFs in Nrf2-deficient mice treated with NFT at 70 mg/kg were significantly greater than those in the control group (Table 2). Increases in G-base substitutions including G:C to T:A or G:C to C:G transversions were observed in Nrf2-deficient mice treated with NFT, although there were no statistically significant differences (Table 4). The results of the Spi– assay are summarized in Table 5. There were no significant changes in Spi– MFs in Nrf2-proficient and Nrf2-deficient mice treated with NFT or NFA at any dose.

Table 2.

Gpt Mutation Frequencies in Kidneys of Nrf2+/+ or Nrf2−/−gpt Delta Mice Treated with NFT or NFA for 13 Weeks

Table 3.

Mutation Spectra in the Kidneys of Nrf2+/+ gpt Delta Mice Treated with NFT or NFA for 13 Weeks

Table 4.

Mutation Spectra in the Kidneys of Nrf2−/− gpt Delta Mice Treated with NFT or NFA for 13 Weeks

Table 5.

Spi– Mutant Frequencies in Kidneys of Nrf2+/+ or Nrf2−/−gpt Delta Mice Treated with NFT or NFA for 13 Weeks

Discussion

Nrf2 plays a crucial role in protection against oxidative stress by transcriptionally upregulating various antioxidant enzymes, including NQO1[12], [13]. Previous studies have shown that Nrf2−/− mice show high sensitivity to various toxicants, including the induction of the oxidative stress response following exposure to acetaminophen, 4-vinylcyclohexene diepoxide, pentachlorophenol, 2-amino-3-methylimidazo[4,5-f]quinoline, ferric nitrilotriacetate, and piperonylbutoxide[14], [15], [16], [17], [18], [19], [20]. Although there were no dose-dependent effects in either genotype, the mRNA expression level of Nqo1 in the kidneys of vehicle-treated Nrf2−/− mice was significantly lower than that of vehicle-treated Nrf2+/+ mice, consistent with the results observed for the protein expression of NQO1. Thus, our results confirmed that Nrf2−/− mice are susceptible to oxidative stress. NFT administration for 13 weeks resulted in a significant increase in 8-OHdG in a dose-dependent manner only in the kidneys of Nrf2−/− mice. Administration of NFA also tended to result in a dose-dependent increase in 8-OHdG in Nrf2−/− mice. These results in the present study suggested that NFT and NFA induced oxidative stress in the kidneys of mice and that NFT might induce severer oxidative stress than NFA.

A significant increase in gpt MFs was observed in the kidneys of NFT-treated Nrf2−/− mice, but not in Nrf2+/+ mice. In NFT-treated Nrf2−/− mice, the frequencies of specific mutations and, in particular, the rates of G:C to T:A and G:C to C:G transversions increased in a dose-dependent manner. These changes in spectra of gpt mutations were consistent with those observed in NFT-treated gpt delta rats[11]. Since guanine bases are susceptible to oxidative modification, the characteristics of the mutation spectra suggest that oxidative stress is involved in NFT-induced genotoxicity. Moreover, 8-OHdG causes G:C to T:A transversions via mispairing with adenine in the course of DNA replication[26], [27]; accordingly, the formation of 8-OHdG may contribute to the G:C to T:A transversions observed in Nrf2−/− mice treated with NFT. Furthermore, NFT failed to induce increases in 8-OHdG in Nrf2+/+ mice, unlike in rats[11], indicating that the sensitivity to oxidative stress is greater in rats than in mice. Considering that NFT does not show carcinogenicity in mice[7], this may explain the difference in NFT carcinogenicity between rats and mice.

Nitro reduction causes oxidative stress in most nitro compounds, including nitrofurans[8], [9], [10]. Nitroreductase induces a one-electron reduction of the nitro group, yielding nitro anion radicals, and the chemical instability increases various ROS, such as superoxide anions and hydroxyl radicals, via its electron-donating ability[28]. ROS generation by nitroreductase is involved in NFT-induced DNA damage or cytotoxicity in rodent livers and lungs[29], [30]. However, our recent report showed that NFA, a constituent compound of NFT with a nitro group, induced a significant increase in the gpt MF, without an elevation in 8-OHdG, in gpt delta rats[11]. In the present study, NFA did not increase MFs of the reporter genes in the kidneys of either genotype, despite the tendencies toward increases in 8-OHdG in NFA-treated Nrf2−/− mice. These results concerning NFA in rats and mice indicated that it is unlikely that oxidative stress is involved in the genotoxicity of NFA; other factors, such as the direct formation of DNA adducts, as observed for other nitrofurans[31], [32], by NFA, likely to contribute to its genotoxicity.

The results of the present study imply that nitro reduction plays a key role in the genotoxicity of NFT. However, our findings indicate the involvement of oxidative DNA damage in genotoxicity in the kidneys of NFT-treated Nrf2−/− mice, but not in the kidneys of NFA-treated Nrf2−/− mice. Side chain interactions may affect the generation of oxidative stress by nitro reduction of the nitro group.

In conclusion, the results of the present study demonstrated that oxidative stress is involved in NFT-induced genotoxicity in mouse kidneys, consistent with previous results in rats, and that oxidative stress was not involved in the genotoxic mechanism of NFA, a constituent compound of NFT with a nitro group. This might be due to the influence by side chains on the generation of oxidative stress by the nitro reduction of the nitro group. The oxidative stress induced by side chain binding should be considered in the development of new nitrofuran compounds.

Disclosure of Potential Conflicts of Interest

The authors declare that they have no competing interests.