Genotoxicity of furan in Big Blue rats

Mutation Research 742 (2012) 72–78

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Genotoxicity of furan in Big Blue rats L. Patrice McDaniel a , Wei Ding a , Vasily N. Dobrovolsky a , Joseph G. Shaddock Jr. a , Roberta A. Mittelstaedt a , Daniel R. Doerge b , Robert H. Heflich a,∗ a b

Division of Genetic and Molecular Toxicology, U.S. Food and Drug Administration/National Center for Toxicological Research, Jefferson, AR 72079, USA Division of Biochemical Toxicology, U.S. Food and Drug Administration/National Center for Toxicological Research, Jefferson, AR 72079, USA

a r t i c l e

i n f o

Article history: Received 27 September 2011 Received in revised form 9 December 2011 Accepted 10 December 2011 Available online 20 December 2011 Keywords: Pig-a mutation Hprt mutation Comet assay Micronucleus assay Cancer mode of action

a b s t r a c t Furan is a multispecies liver carcinogen whose cancer mode of action (MOA) is unclear. A major metabolite of furan is a direct acting mutagen; however, it is not known if genotoxicity is a key step in the tumors that result from exposure to furan. In order to address this question, transgenic Big Blue rats were treated by gavage five times a week for 8 weeks with two concentrations of furan used in cancer bioassays (2 and 8 mg/kg), and with two higher concentrations (16 and 30 mg/kg). Peripheral blood samples taken 24 h after the 5th dose (1 week of dosing) were used to assay for micronucleus (MN) frequency in normochromatic erythrocytes (NCEs) and reticulocytes (RETs), and Pig-a gene mutation in total red blood cells (RBCs). 24 h after the last dose of the 8-week treatment schedule, the rats were euthanized, and their tissues were used to perform NCE and RET MN assays, the Pig-a RBC assay, Pig-a and Hprt lymphocyte gene mutation assays, the liver cII transgene mutation assay, and the liver Comet assay. The responses in the MN assays conducted at both sampling times, and all the gene mutation assays, were uniformly negative; however, the Comet assay was positive for the induction of liver DNA damage. As the positive responses in the Comet assay were seen only with doses in excess of the cancer bioassay doses, and at least one of these doses (30 mg/kg) produced toxicity in the liver, the overall findings from the study are consistent with furan having a predominantly nongenotoxic MOA for cancer. Published by Elsevier B.V.

1. Introduction Furan is a small heterocycle (C4 H4 O, MW 68.08) that, as a pure substance, is a colorless, relatively volatile liquid (boiling point, 31.4 ◦ C). Furan is used industrially as a solvent and as an intermediate in chemical syntheses with little indication of worker exposure [1]. Furan, however, also is generated as a combustion product, and it is released into the air in the gas phase of cigarette smoke. Importantly, furan has been detected in many common cooked foods, including jarred baby foods and canned infant formula, where concentrations over 100 ppb have been detected [2,3]. This possibility for human exposure, especially for infants, is a potential health risk since furan is a relatively potent multispecies carcinogen, producing liver tumors in both mice and rats [4]. Based on its rodent carcinogenicity, the International Agency for Research on Cancer has classified furan as possibly carcinogenic to humans (Group 2B) [1].

∗ Corresponding author at: Division of Genetic and Molecular Toxicology, HFT120, U.S. FDA/NCTR, 3900 NCTR Rd., Jefferson, AR 72079, USA. Tel.: +1 870 543 7493; fax: +1 870 543 7393. E-mail address: robert.hefl[email protected] (R.H. Heflich). 1383-5718/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.mrgentox.2011.12.011

A number of studies have been conducted to understand the mechanisms involved in the carcinogenicity of furan in rodents; however, it is not yet clear whether furan acts primarily through a nongenotoxic or genotoxic mode of action (MOA). Furan is metabolized by hepatic cytochromes P450 (CYPs), predominantly CYP2E1 [5], to a reactive intermediate, cis-2-butene-1,4-dial (BDA) [6,7]. BDA forms adducts with DNA bases in vitro [8,9] and is a direct-acting genotoxin in the Salmonella reversion assay and in mammalian cells [10,11], suggesting that the tumors induced by furan could be generated as a result of its genotoxicity. Agents that require CYP2E1 metabolism, however, are often difficult to detect in standard in vitro genotoxicity assays. Furthermore, BDA is extremely reactive with many biologically important nucleophiles, including amine and thiol groups [12], which renders it highly cytotoxic, complicating the analysis of its genotoxicity [11]. Perhaps because of these factors, furan itself has tested negative in the Salmonella reversion assay [4,13], and in most reports, it also has been negative or weakly positive in standard in vitro mammalian cell assays (sister-chromatid exchange (SCE), chromosome aberration, micronucleus (MN), Comet, mouse lymphoma gene mutation). Positive responses in the mammalian cell assays typically were detected only at very high concentrations, and in some cases, using protocols that are no longer considered acceptable [4,11,14–17].

L.P. McDaniel et al. / Mutation Research 742 (2012) 72–78

In addition, furan genotoxicity was only marginally increased in transgenic V79 cells expressing human CYP2E1 [16]. The genotoxicity of carcinogens that are metabolized primarily by CYP2E1 is often more readily detected by in vivo than by in vitro assays [18]. For instance, acrylamide, which is metabolized by CYP2E1 to its epoxide, glycidamide, and is only weakly genotoxic in vitro, produces positive responses in a variety of in vivo genetic toxicity tests in both mice and rats [19,20]. Mixed results have been reported from the in vivo genetic toxicology testing performed on furan, however. In work conducted in conjunction with National Toxicology Program (NTP) cancer bioassays, furan was positive for chromosome aberration but negative for SCE, both conducted in mouse bone marrow cells [4], and negative for unscheduled DNA synthesis in hepatocytes from furan-exposed rats [21]. Also, oncogene patterns differed in a small number of liver tumors from furan-treated and control mice, indicating that furan genotoxicity could be involved in tumor induction [22]. In two more recent reports on mice treated subchronically with up to 15 mg/kg/day furan, splenocytes were positive for MN and ␥-H2AX focus induction, but negative for DNA damage by the Comet assay [23], while liver was negative for ␥-H2AX induction and DNA damage by the Comet assay [24]. Liver DNA damage was detected by Comet in mice given a single dose of 250 mg/kg furan [24], but treatment of mice with acute doses of up to 300 mg/kg did not increase MN frequency in peripheral blood reticulocytes (RETs) [17]. Finally, there was no liver DNA binding in rats dosed orally on 8 consecutive days with 8 mg/kg of 14 C-furan [25], but the sensitivity of this analysis was limited by the low specific activity of the radiolabel. There is evidence, however, that furan may damage liver DNA indirectly, through the generation of reactive oxygen species [26]. The available data, therefore, indicate that furan has genotoxic potential. Whether or not that potential manifests itself in a manner that contributes to its carcinogenicity remains an open question. Because of the limitations of in vitro assays for assessing the genotoxicity of furan, in vivo data are necessary for answering this question, and there is very little known about the genotoxicity of furan in rats. Thus, in the present study, we evaluated the genotoxicity of furan in transgenic Big Blue rats using subchronic treatments modeled on the dosing protocol used by the NTP that resulted in high frequencies of hepatocarcinomas and cholangiocarcinomas [4]. The potential of furan to produce systemic genotoxicity was evaluated using the peripheral blood MN assay, the Hprt lymphocyte gene mutation assay, and the Pig-a lymphocyte and peripheral red blood cell (RBC) gene mutation assays. In addition, we have evaluated genotoxicity in the major target tissue for furan carcinogenicity by measuring liver cII mutant frequency in the Big Blue transgene and liver DNA damage using the in vivo Comet assay. 2. Materials and methods 2.1. Chemicals and reagents Furan (CAS 110-00-9) was purchased from Aldrich Chemical (Milwaukee, WI), redistilled before use, and stored at −20 ◦ C under nitrogen in sealed septum vials. Fresh solutions of furan were prepared weekly in corn oil (CAS 8001-30-7; Sigma, St. Louis, MO). RecoverEase DNA Isolation Kits and the Select-cII Mutation Detection System for Big Blue Rodents (contains Transpack packaging extract, and Escherichia coli G1250) were obtained from Stratagene (La Jolla, CA). Rat MicroFlowPlus kits were purchased from Litron Laboratories (Rochester, NY), while Lympholyte-R was from Cedarlane Laboratories (Burlington, NC).

2.2. Animals and treatments The study was conducted with the approval and oversight of the NCTR Institutional Animal Care and Use Committee. Male Big Blue transgenic rats were obtained as weanlings from Taconic Farms (Germantown, NY), housed two per cage, and aged to 50 days old before dosing. Rats were fed 5LG6 rodent diet (Purina Mills, Brentwood, MO) and provided drinking water ad libitum. 5LG6 diet was analyzed by GC/MS and found to have a furan content of <10 ppb.

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Rats were weight ranked and, using this information, five treatment groups were formed, each containing 6 rats. Rats were weighed daily and dosed by gavage 5 days per week for 8 weeks with corn oil (vehicle control), or with 2, 8, 16, or 30 mg/kg furan in corn oil. In keeping with the goal of reducing animal use, and because the investigators have extensive experience with the assays used in this study, positive control groups were not employed for the various assays. The volume of the doses was 5 ml/kg body weight of the rats. The 2 and 8 mg/kg doses were the low and high doses used in the NTP cancer bioassay [4], while the 30 mg/kg dose was tolerated in previous subchronic rat dosing studies [4,21,26,27]. The dosing was conducted in a staged manner, with 2 rats from each group beginning the dosing regimen at weekly intervals. On Day 7 of dosing (24 h after the 5th dose for Week 1), a 0.1 ml blood sample was taken from the tail vein and diluted 1:3 with 500 units/ml heparin. 180 ␮l of heparinized blood were fixed in 2 ml of −80 ◦ C methanol and stored at −80 ◦ C for use in MN assays; the remainder of the blood was used for the RBC Pig-a assay. The rats were sacrificed 24 h after the last dose (Day 56) by exposure to gaseous CO2 . Spleens were removed aseptically, crushed, and splenocytes purified by centrifugation on Lympholyte-R for use in Hprt and Pig-a lymphocyte assays. Blood was collected via cardiac puncture and processed for MN and RBC Pig-a assays as described for Day 7 samples. Liver and other tissues were immediately removed, and a thin slice of liver was used for the Comet assay. The remainder of the liver and other tissues were frozen in liquid nitrogen and stored at −80 ◦ C for use in transgene mutation assays. 2.3. Pig-a RBC gene mutation assay The RBC Pig-a assay was performed as described previously [28], with refinement of the gating strategy for the enumeration of CD59-deficient RBCs similar to the human PIG-A assay outlined in Dobrovolsky et al. [29]. 5 ␮l of heparinized blood were labeled in a mixture containing 200 ␮l Mg2+ , Ca2+ -free phosphate-buffered saline (PBS; Invitrogen, Carlsbad, CA), 0.25 ␮l of 0.5 mg/ml biotin-conjugated HIS49 antibody (BD Pharmingen, San Jose, CA), and 2 ␮l of 0.5 mg/ml FITC-conjugated antirat CD59 antibody (BD Pharmingen). After incubation for 30 min in the dark at room temperature, 1 ␮l of 0.2 mg/ml APC-conjugated Streptavidin (BD Pharmingen) was added for fluorescent labeling of HIS49-reactive epitopes. After an additional 30 min incubation in the dark at room temperature, the labeled cells were centrifuged for 3 min at 300 × g, the cell pellet was resuspended in 1 ml of flow sheath fluid, and the suspension was filtered through the cell-strainer cap into 5 ml polystyrene round-bottomed tubes (BD FalconTM , Franklin Lakes, NJ). In parallel, two flow cytometry standards were prepared: one was prepared from control blood labeled with biotin-HIS49/APC-Streptavidin (1-color mutant-mimic standard), and the unstained standard was prepared from control blood mixed with APC-Streptavidin only (without using primary antibodies). The analysis was performed on a FACSAria I flow cytometer (BD Biosciences, San Jose, CA) equipped with 488 nm blue and 635 nm red lasers and the default filter set. All fluorescence and light scatter parameters were measured in area mode. Single cells were roughly outlined on a forward vs. side scatter cytogram; these cells were further analyzed on an APC fluorescence histogram, where the major peak representing the fraction of single-cell HIS49-positive RBCs was gated. Single-cell RBCs were further analyzed for CD59 expression. The gate for CD59-deficient mutants was identified using the 1-color-stained mutant-mimic standard. The mutant gate contained 98% of the single-cell RBCs from the APC-only labeled standard. At least 1 × 106 total events were processed (of which 7 to 8 × 105 were single-cell RBCs) in order to identify the frequencies of CD59-deficient (Pig-a mutant) RBCs in 2-color-labeled experimental samples. 2.4. Micronucleus assay Methanol-fixed blood samples were assayed for MN frequency using a FACSort flow cytometer (BD Biosciences) and Rat MicroflowPlus Kits. The protocol described by Mei et al. [20] was followed. The stopping gate was set to analyze a total of 20,000 RETs and data on %RETs, percent micronucleated RETs (%MN-RETs), and percent micronucleated normochromatic erythrocytes (%MN-NCEs) were collected. Positive and negative controls provided with the MicroFlowPlus Kit were run as controls for the flow cytometric analysis. 2.5. Pig-a lymphocyte gene mutation assay Gradient purified mononuclear cells from approx. 25% of the spleen from a subset of the animals (4–6 per treatment group) were resuspended in 0.5 ml of PBS and mixed with 5 ml of 1× RBC lysis buffer (BioLegend, San Diego, CA) in a 15 ml tube in order to eliminate the remaining contaminating RBCs. After a 5-min incubation on ice, 10 ml of PBS were added to the RBC-depleted samples, and the enriched white blood cells were pelleted by centrifugation for 5 min at 500 × g. The off-white cell pellets were washed again in 10 ml PBS and resuspended in 0.4 ml of PBS. 100 ␮l of these purified white blood cells were mixed with 100 ␮l PBS, 3 ␮l of 0.2 mg/ml APC-conjugated anti-rat TCR ␣/␤ (BioLegend) and 3 ␮l of 0.2 mg/ml PE-conjugated anti-rat CD48 (BioLegend). The flow cytometry standards were an unstained sample from a control rat, and a control sample stained with APC anti-TCR only (mutantmimic standard). The labeling proceeded for 30 min at 4 ◦ C, and then the cells were centrifuged 3 min at 300 × g, resuspended in 1 ml of flow sheath fluid, and filtered

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through the cap sieve of a 5 ml polystyrene round-bottomed tube. Analysis for CD48deficient mutant T-lymphocytes was performed on the 2-laser FACSAria I described above. The approximate region containing lymphocytes was gated on a forward/side light scatter cytogram; these cells were further analyzed on an APC histogram, where TCR-positive cells produced a distinct peak consisting of bright fluorescent cells well separated from the unstained TCR-negative cells. The TCR-positive T-lymphocytes were analyzed for the frequency of CD48-deficient (PE dim) Pig-a mutants. The gate for mutant T-cells was identified using the mutant-mimic standard; the mutant gate contained 98% of TCR-positive cells from this 1-color-stained sample. 2.6. Hprt lymphocyte assay The assay was modified from the assay for mouse spleen cells described by Dobrovolsky et al. [30] and as described for rats by Mei et al. [20]. In brief, gradient purified spleen lymphocytes were primed overnight with concanavalin A. The lymphocytes then were used to establish two sets of cultures in 96-well round-bottom microtiter plates, one containing four primed lymphocytes plus 4 × 104 irradiated feeder lymphocytes per well in growth medium and one containing 4 × 104 primed lymphocytes per well in growth medium supplemented with 2 ␮g/ml 6-thioguanine (Sigma). After incubating the plates for 10–12 days, the wells were scored for colony formation using a fluorescent viability stain as described previously [31]. The cloning efficiencies and the frequency of 6-thioguanine-resistant (Hprt mutant) T lymphocytes were calculated using Poisson statistics. 2.7. Liver Comet assay Single cell suspensions were prepared from thin slices of liver by mincing with sharp scissors; the minced tissue was filtered through a 40 ␮m cell strainer (Fisher Scientific, Pittsburg, PA). The standard alkaline Comet assay was performed using established methods [32,33], with appropriate modifications. Briefly, the single cell suspensions were mixed with 1% low melting-point agarose (LMP) in PBS at 37 ◦ C and applied to microscope slides (Fisher Scientific) previously coated with 1% agarose. After the slides were held at 4 ◦ C for 30 min to solidify the LMP, they were placed in freshly prepared lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, adjusted to pH 10 with NaOH, and with 10% dimethylsulfoxide and 1% Triton X-100 added just before use) and stored at 4 ◦ C overnight. The slides then were transferred into a chilled alkaline solution (300 mM NaOH, 1 mM EDTA, pH 13). After unwinding for 40 min, electrophoresis was performed in the same solution at 4 ◦ C in the dark for 30 min at 20 V and ∼300 mA. The slides then were washed with neutralizing buffer (0.4 M Tris–HCl, pH 7.5) 3× for 5 min each wash, washed with ice cold 100% ethanol for 5 min, and dried overnight. Prior to scoring, the slides were stained with SYBR Gold (Invitrogen; 1:10,000 dilution in TBE buffer). Two slides were scored for each liver, with at least 100 cells selected randomly from each slide. The slides were scored for %DNA in the Tail as the measure of DNA damage using a system consisting of a Nikon 501 fluorescent microscope and Comet IV digital imaging software (Perceptive Instruments, Wiltshire, UK). 2.8. cII mutant assay High-molecular-weight genomic DNA was extracted from rat liver using the RecoverEase DNA Isolation Kit. The packaging of the phage, plating the packaged DNA samples, and determination of mutants were performed according to the manufacturer’s instructions for the Select-cII Mutation Detection System for Big Blue Rodents. In most cases the assay was repeated until a minimum of 2 × 105 plaqueforming units from each sample was examined for mutation. 2.9. Statistical analyses The animal was the experimental unit for all statistical analyses. Animal weight data were analyzed by repeated measures analysis of variance (ANOVA) followed by the Holm–Sidak method for pairwise comparisons between the treatment groups and the vehicle control. Depending on whether or not they were normally distributed, genotoxicity data were analyzed by ANOVA or ANOVA on ranks, followed by pairwise comparisons of responses in the treatment groups to the vehicle control group using Dunnett’s test or Dunn’s test. Square root and log data transformations did not consistently normalize the data and thus were not employed. All tests were two-sided and P < 0.05 was used to detect significant differences.

3. Results Groups of 6 male Big Blue rats were treated with up to 30 mg/kg furan 5 days a week for 8 weeks. Body weight measurements made over the treatment period indicated that only the rats treated with the high dose of furan had a decrease in body weight gain; at the time of sacrifice, the rats dosed with 30 mg/kg furan weighed 82% of the weight of rats in the vehicle control group, and had 56% of the weight gain of the vehicle control rats over the 8 weeks dosing

period (Table 1). Also, at necropsy, mottled, hard, strawberrycolored livers were observed in the rats dosed with 30 mg/kg furan. After 1 week of dosing, RET and NCE MN assays and the Piga RBC gene mutation assay were conducted on peripheral blood samples from the animals (Table 2). Furan dosing had no significant effect on the frequency of micronucleated RETs or NCEs or on the frequency of Pig-a RBC mutants (as measured by the frequency of CD59-negative RBCs). There was a small dose-related decrease in the %RETs in the blood samples, but the decrease was not great enough to reach statistical significance. Table 3 shows the results of endogenous gene mutation assays, the liver Comet assay, and MN assays conducted at terminal sacrifice after 8 weeks of dosing with furan. As was the case with the Week 1 measurements, furan treatment had no effect on either the RBC Pig-a mutant frequency or on the MN endpoints. There was a significant effect on %RET that manifested itself as an increase in the high dose furan group. Pig-a and Hprt gene mutation assays were performed on spleen lymphocytes from the animals, and neither of these assays showed a treatment effect. In contrast to these largely negative responses, significant increases in DNA damage were noted in liver Comet assays conducted on rats treated with 16 mg/kg and 30 mg/kg furan. Finally, liver cII mutant frequency was evaluated using the Big Blue rat transgene (Table 4). With or without the outlier mutant frequency of Rat 31 included, furan treatment had no significant effect on liver cII mutant frequency.

4. Discussion Furan is a relatively potent, multispecies carcinogen that is found in food and other environmental sources. Thus, furan presents a potential health risk for humans and consequently a more complete toxicological assessment of furan has been initiated both in Europe and by the U.S. Food and Drug Administration [34–36]. A major part of this assessment involves determining furan’s MOA for cancer in rodents, since agents with a genotoxic MOA are viewed as having an increased potential for risk at low doses (e.g., [37,38]). Also, the risks associated with low levels of genotoxic agents are believed to be increased when exposures involve children, as is the case with furan. Groups investigating the carcinogenicity of furan have reached different conclusions, or mixed conclusions, regarding its cancer MOA (e.g., [12,21,23,24,39–41]). Furan is clearly toxic to liver, causing cell death followed by regenerative cell proliferation [4,21,27,39,42,43], which could be the basis for a nongenotoxic MOA for cancer in chronically exposed animals. No histopathology was conducted in our study, but some observations may be relevant to furan’s toxicity. The rats dosed with 2 and 8 mg/kg furan (two doses that were used in the NTP cancer bioassay) had higher weight gains than the vehicle control group (although not significantly higher), which is at least consistent with furan promoting cellular proliferation as documented by the NTP [4]. By contrast, the weight gain for rats dosed with 30 mg/kg furan was significantly less than the control, and visual inspection indicated that the liver morphology in these rats was altered, consistent with the toxicity of the agent. Note that liver weight may have been more sensitive than body weight to any proliferative effect of furan, but this measurement was not made. In support of a genotoxic MOA, furan is converted by liver enzymes to a genotoxic metabolite, BDA, indicating that a genotoxic MOA is at least possible. A complication is that furan is a small molecule that is metabolized by CYP2E1, and the genotoxicity of these types of compounds (e.g., acrylamide, ethyl carbamate) is difficult to detect in vitro [18]. So weak genotoxic responses for furan, at least in vitro, are not unexpected. However, published data,

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Table 1 Weight of Big Blue F344 rats during 8 weeks of dosing with furan. Data are the mean ± SD of weights of 5-6 rats per group (one of the vehicle control rats was lost during Week 3). Dose (mg/kg)

0 (vehicle control) 2 8 16 30 RM ANOVA

Rat weight in gm (mean ± SD) Week 0

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

181.5 ± 52.6 187.6 ± 49.4 190.4 ± 33.9 187.7 ± 35.2 188.7 ± 34.3 P = 0.797

207.4 ± 49.0 217.4 ± 43.6 219.1 ± 27.0 203.4 ± 31.7 194.9 ± 26.3 P = 0.047

227.4 ± 46.8 242.9 ± 39.8 244.4 ± 23.8 227.6 ± 29.2 209.3 ± 25.7 P = 0.004

250.2 ± 43.8 266.0 ± 37.7 267.0 ± 20.7 250.0 ± 26.7 227.1 ± 21.1 P < 0.001

271.4 ± 41.0 287.1 ± 35.1 289.1 ± 18.3 269.1 ± 24.3 239.5 ± 18.1* P < 0.001

288.4 ± 37.8 306.6 ± 32.1 306.1 ± 17.4 284.8 ± 22.8 250.7 ± 16.7* P < 0.001

305.3 ± 36.6 325.3 ± 34.1 323.6 ± 17.5 300.5 ± 22.6 260.1 ± 15.2* P < 0.001

319.8 ± 38.1 339.9 ± 32.7 335.7 ± 14.4 313.9 ± 23.7 267.2 ± 13.0* P < 0.001

332.9 ± 37.0 352.1 ± 34.3 349.5 ± 13.1 327.3 ± 20.0 272.8 ± 11.6* P < 0.001

RM ANOVA, repeated measures analysis of variance. * Significantly different from weights of vehicle control (Holm–Sidak method), P < 0.05. Table 2 Pig-a red blood cell (RBC) mutant frequency and micronucleus (MN) frequency in Big Blue rats following one week exposure to furan. Rats were given five daily doses over a 7 day period and assays conducted one day after the final dose (Day 7). Furan dose (mg/kg)

N

Pig-a mutants (×10−6 RBCs (±SD))

0 2 8 16 30 ANOVA (ANOVA on ranks) Litron negative MN controla Litron positive MN controla T-test

6 6 6 6 6

1.63 ± 1.72 0.38 ± 0.94 0.40 ± 0.62 0.20 ± 0.49 1.00 ± 1.61 P = 0.17

a

3 3

MN measurements (±SD) %RETs

%MN-RETs

%MN-NCEs

2.08 ± 0.43 2.35 ± 0.41 2.17 ± 0.19 1.68 ± 0.23 1.47 ± 0.14 P = 0.09 1.69 ± 0.03 0.97 ± 0.01 P < 0.001

0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.05 ± 0.00 0.05 ± 0.01 P = 0.31 0.10 ± 0.01 0.68 ± 0.03 P < 0.001

0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 P = 1.00 0.00 ± 0.00 0.01 ± 0.00 P = 0.2

Controls included with the assay kit (see Section 2).

mostly from mouse studies, indicate that the in vivo genotoxicity of furan is also weak and inconsistent (see Introduction), and the data from our study in rats confirm this general finding. Even after 8 weeks treatment with doses of furan that induced cancer after lifetime exposure and two even higher doses, including one that was clearly hepatotoxic to the rats, all of the gene mutation and MN assays that we conducted were negative. Cholangiofibrosis, believed to be a precursor lesion of cholangiocarcinomas, develops within the first month of furan treatment [27]. Therefore, if furan has a mutagenic MOA, 8 weeks of treatment should be more than adequate for detecting its genotoxicity [44]. The genotoxic metabolite of furan, however, is likely generated in the liver, and, because of its very high reactivity with glutathione and other hepatocyte constituents, may not reach the bone marrow where micronuclei and the mutations measured in the Pig-a and Hprt

assays are formed [45–48]. Despite the induction of hepatocellular adenoma/carcinoma and cholangiocarcinoma by chronic exposure to the lower furan doses used in our study, liver cII assays were also negative in furan-treated rats. It is possible that the sampling times were not optimum for detecting the maximum induced responses in the gene mutation assays that we employed. For instance, maximum Hprt lymphocyte and Pig-a RBC responses in rats are typically detected 4–6 weeks after dosing [47,49]. Mutant manifestation times for in vivo transgenic systems vary with the tissue being sampled but can be as long as several months [50]. These relatively long manifestation times for the gene mutation endpoints contrast with optimum manifestation times of hours to days for the Comet and MN assays [33,51]. In our study, the prolonged (8-week) treatment schedule and sampling 24 h after the last dose of the treatment was intended to be a

Table 3 Endogenous gene mutant frequencies, liver DNA damage measured by the Comet assay, and micronucleus (MN) frequency in Big Blue rats treated 5 days per week for 8 weeks with furan. Assays were conducted on rats sacrificed 1 day following the final dose (Day 56). Furan dose (mg/kg)

0 2 8 16 30 ANOVA/ANOVA on ranks Litron negative MN controla Litron positive MN controla a * **

N

Liver Comet measurements

MN measurements (±SD)

% DNA in Tail (±SD)

%RETs

%MN-RETs

%MN-NCEs

6.92 ± 0.45 8.05 ± 1.05 7.11 ± 2.04 11.82 ± 1.44* 17.62 ± 3.86* P < 0.001

1.05 ± 0.10 1.37 ± 0.18 0.99 ± 0.03 1.22 ± 0.03 1.48 ± 0.09** P = 0.004

0.03 ± 0.01 0.03 ± 0.01 0.05 ± 0.01 0.04 ± 0.01 0.04 ± 0.01 P = 0.69

0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 P = 0.06

1

5.12

0.12

0.02

1

0.86

1.71

0.03

5 5-6 4-6 6 6

Pig-a mutants (×10−6 RBCs (±SD))

2.48 ± 1.97 1.88 ± 1.85 2.38 ± 1.39 1.83 ± 1.51 2.65 ± 1.13 P = 0.86

Pig-a mutants (×10−6 lymphocytes (±SD))

12.02 ± 8.37 6.66 ± 4.08 12.48 ± 1.65 12.22 ± 5.85 12.77 ± 12.36 P = 0.71

Hprt mutants (×10−6 lymphocytes (±SD))

3.98 ± 1.65 2.85 ± 0.94 4.48 ± 2.62 4.25 ± 1.28 1.77 ± 1.51 P = 0.06

Controls included with the assay kit (see Section 2.1). Significantly different compared with vehicle control (Dunn’s test), 0 < 0.05. Significantly different compared with vehicle control (Dunnett’s test), 0 < 0.05.

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Table 4 Liver cII mutant frequencies in Big Blue rats treated 5 days per week for 8 weeks with furan. Assays were conducted with rats sacrificed 1 day following the last dose (Day 56). Furan dose (mg/kg)

Rat number

Number of packagings

Total phages screened (×103 )

Total mutants observed

Mutant frequency (×10−6 )

0

1 2 3 4 5 6a

3 5 4 3 3

391.3 207.7 319.0 240.0 341.3

26 6 15 12 11

66.5 28.9 47.0 50.0 32.2

11 12 13 14 15 16

5 3 2 5 3 2

296.3 200.0 218.7 246.0 204.0 251.7

15 10 10 20 7 8

21 22 23 24 25 26

3 3 4 6 5 2

217.3 207.7 235.0 192.0 270.0 207.7

7 9 6 8 16 5

31 32 33 34 35 36

3 3 5 4 5 6

213.7 159.0 279.3 308.3 266.7 288.7

40 6 11 11 13 15

41 42 43 44 45 46

4 4 3 4 5 6

219.7 306.0 54.3 315.0 336.0 224.0

12 24 3 7 16 11

Group mean ± SD 2

Group mean ± SD 8

Group mean ± SD 16

Group mean ± SD 30

Group mean ± SD ANOVA on ranks ANOVAb a b

44.9 ± 15.1 50.6 50.0 45.7 81.3 34.3 31.8 48.9 ± 17.1 32.2 43.3 25.5 41.7 59.3 24.1 37.7 ± 13.3 187.2 37.7 39.3 35.7 48.7 52.0 66.8 ± 59.4 42.7 ± 7.2b 54.6 78.4 55.2 22.2 47.6 49.1 51.2 ± 18.0 P = 0.565 P = 0.573

Rat died during dosing. With outlier data from Rat 31 removed.

reasonable compromise for the requirements of the various assays that were conducted. The only assay yielding a positive response in furan-treated rats was the liver Comet assay; however, the interpretation of this response is not straightforward. First, the positive response was detected at the two highest furan doses, both greater than the doses used in the NTP cancer bioassay, and the 30 mg/kg dose was clearly toxic to the liver. Although toxicity is considered a confounder for the in vitro Comet assay [33], it is not clear whether or not toxicity produces false positives in the in vivo assay [51]. Second, the sampling time for the Comet assay (24 h after the last dose) is at the upper limit recommended for detecting DNA damage in the Comet assay [33,52]. It could be that earlier sampling times may have resulted in a more robust response, with positive responses at lower doses. In partial support of this possibility, preliminary Comet experiments indicate that DNA damage is more readily detected at 1 h than at 24 h after an acute treatment with 16 mg/kg furan [53]. Unfortunately conducting the liver Comet assay typically involves killing the animals, and sufficient animals were not available in the present study for employing multiple sampling times. Therefore, there are two potential problems with our Comet assay responses: toxicity may have been a confounder for the positive Comet responses that we detected, and we may have detected positive responses at lower, bioassay doses had we used another sampling time.

In conclusion, transgenic rats were treated for 8 weeks with four doses of furan, two doses used in the NTP cancer bioassay (2 and 8 mg/kg/day) and two higher doses, one of which (30 mg/kg/day) that produced toxicity in the rats. Negative responses were detected in the MN assay and in gene mutation assays conducted with the lymphocyte Hprt and Pig-a genes, the erythrocyte Pig-a gene, and with the liver cII transgene. Positive responses for DNA damage were detected at the two highest furan doses in the liver Comet assay. The data generally are consistent with a nongenotoxic MOA for the carcinogenicity of furan, but will have to be combined in a weight-of-evidence analysis to make a final determination.

Acknowledgements and disclaimer This study was supported by Interagency Agreements 22407-007 and Y1ES1027 between the National Institute for Environmental Health Sciences/National Toxicology Program and the U.S. Food and Drug Administration/National Center for Toxicological Research. WD was supported by a postdoctoral appointment from the Oak Ridge Institute for Science and Education. The authors are grateful for the help provided by Michelle Bishop, Lascelles Lyn-Cook, Mason Pearce, Lynda McGarrity, and Drs. Ben Aidoo and Manju Manjanatha. The views presented in this communication are not necessarily those of the U.S. Food and Drug Administration.

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