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Assessment of DNA Binding and Oxidative DNA Damage by Acrylonitrile in Two Rat Target Tissues of Carcinogenicity: Implications for the Mechanism of Action Gary M. Williams,* Tetyana Kobets, Jian-Dong Duan, and Michael J. Iatropoulos Chemical Safety Program, Department of Pathology, New York Medical College, Valhalla, New York 10595, United States ABSTRACT: Exposure to acrylonitrile induces formation of tumors at multiple sites in rats, with females being more sensitive. The present study assessed possible mechanisms of acrylonitrile tumorigenicity, covalent DNA binding, DNA breakage, and oxidative DNA damage, in two target tissues, the brain and Zymbal’s glands, of sensitive female Fischer (F344) and Sprague−Dawley (SD) rats. One group received acrylonitrile in drinking water at 100 ppm for 28 days. Two other groups were administered either acrylonitrile in drinking water at 100 ppm or drinking water alone for 27 days, followed by a single oral gavage dose of 11 mg/kg bw 14C-acrylonitrile on day 28. A positive control group received a single dose of 5 mg/kg bw of 7-14C-benzo[a]pyrene, on day 27 following the administration of drinking water for 26 days. Using liquid scintillation counting, no association of radiolabeled acrylonitrile with brain DNA was found. In accelerator mass spectrometry analysis, the association of 14C of acrylonitrile with DNA in brains was detected and was similar in both strains, which may reflect acrylonitrile binding to protein as well as to DNA. Nucleotide 32P-postlabeling assay analysis of brain samples from rats of both strains yielded no evidence of acrylonitrile DNA adducts. Negative conventional comet assay results indicate the absence of direct DNA strand breaks in the brain and Zymbal’s gland in both strains of rats dosed with acrylonitrile. In both rat strains, positive results in an enhanced comet assay were found only in brain samples digested with formamidopyrimidineDNA glycosylase but not with human 8-hydroxyguanine-DNA glycosylase, indicating possible oxidative DNA damage, other than 8-oxodG formation. In conclusion, definitive evidence of DNA binding of acrylonitrile in the brain and Zymbal’s gland was not obtained under the test conditions. A role for oxidative stress in tumorigenesis in the brain but not Zymbal’s gland may exist.

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INTRODUCTION Chronic inhalation or oral administration of acrylonitrile, a chemical intermediate widely used in the manufacture of acrylic fiber and plastics, has produced tumors in various tissues in two strains of rats, Fischer 344 (F344) and Sprague−Dawley (SD),1−4 and one mouse strain, B6C3F1.5 In rats, acrylonitrile primarily causes the formation of microgliomas, Zymbal’s gland carcinomas and tumors of the forestomach in male and female rats and mammary gland in females, while in mice commonly affected sites of acrylonitrile carcinogenicity are forestomach (squamous cell carcinoma) and Harderian glands (adenoma and carcinoma) in both sexes, while lungs (adenoma and carcinoma) and ovary (granulosa-cell tumors) tumors may relate to acrylonitrile exposure.6−10 Immunohistochemical evaluation of the acrylonitrile-induced brain tumors in rats confirmed that all neoplasms were microglial in origin, rather than astrocytomas.7 On the basis of the sufficient evidence of carcinogenicity in experimental animals, acrylonitrile was classified as “reasonably anticipated to be a human carcinogen” by the National Toxicology Program8 and as “possibly carcinogenic to humans” by the International Agency for Research on Cancer.10 Initial indications of an increased cancer risk among workers exposed to acrylonitrile, however, were not confirmed by subsequent, © 2017 American Chemical Society

more informative studies. On the basis of the evidence from several cohort studies, the International Agency for Research on Cancer concluded that there was no increased risk of cancer incidence in workers exposed to acrylonitrile.10 Taking into consideration the cancer classifications for acrylonitrile, regulatory authorities are inclined to develop estimates of human cancer risk associated with low levels of acrylonitrile exposure. Understanding the mode of action of acrylonitrile in producing rodent tumors, particularly in the brains of rats, could contribute to the risk assessment. Several in vitro and in vivo studies intended to elucidate the mechanisms of acrylonitrile carcinogenicity have been reported. In vitro, acrylonitrile was reported to be weakly mutagenic only in the presence of metabolic activation, as detected by reverse mutation assays in several strains of Salmonella typhimurium and in TK6 human lymphoblasts.9,11,12 These positive findings were attributed to the activity of the reactive epoxide, 2cyanoethylene oxide (CEO), formed by acrylonitrile bioactivation by CYP 450.11−14 CEO is reported to have a high binding affinity to DNA and protein in vitro.13,15−18 Additionally, it was found to be mutagenic in human lymphoblastoid cells.19 Received: April 21, 2017 Published: June 14, 2017 1470

DOI: 10.1021/acs.chemrestox.7b00105 Chem. Res. Toxicol. 2017, 30, 1470−1480

Article

Chemical Research in Toxicology

deoxyribonucleotides, rather than the formation of DNA adducts.40 In other more rigorous studies, alkylation of DNA in the rat liver was not observed above the detection limit of one base in 3.5 × 105,21 and alkylation of DNA in the rat brain was not detected above 1 adduct per 106 of normal nucleotides.34 These studies, however, used low specific activity 14C-acrylonitrile in conjunction with liquid scintillation counting (LSC), and thus, the sensitivity was limited. Review of the conflicting evidence for DNA adduct formation in the brain led to the conclusion that in studies reporting DNA adducts, the association was most likely due to contamination with acrylonitrile bound to the residual protein.14 Emerging evidence, together with the lack of definitive data confirming the DNA-binding ability of acrylonitrile in the brain, suggests that the mechanisms involved in acrylonitrile carcinogenicity could result from indirect genotoxicity. Namely, the ability of acrylonitrile to induce oxidative DNA damage, as evidenced by the formation of 8-oxodeoxyguanosine (8-oxodG) and consequent inflammatory response in the brain tissue of rats exposed to acrylonitrile, has been reported.43−47 On the basis of the findings of skin and eye irritation tests in rabbits and evidence of skin irritation in workers who had direct exposure to the compound, acrylonitrile is also considered to be a strong irritant,9,10 which could also contribute to its carcinogenicity since inflammation is linked to carcinogenesis.48 The purpose of the present research was to rigorously assess potential direct and indirect DNA damage by acrylonitrile in the brain and Zymbal’s glands, both target tissues of female F344 and SD rats. This was done by the determination of DNA binding of acrylonitrile in brain tissue using LSC and accelerator mass spectrometry (AMS);49 formation of DNA adducts in brain samples using the nucleotide 32P-postlabeling (NPL) assay;50,51 and assessment of DNA strand breaks and oxidative DNA damage using conventional52−54 and enhanced comet assays.55 For the DNA binding and adduct measurements, benzo[a]pyrene was used as a comparator. Although benzo[a]pyrene is not carcinogenic to the rat brain,56 14C-benzo[a]pyrene forms DNA adducts in several rat tissues, including the brain,57 which have been well characterized and detected with high sensitivity by the NPL assay.58 We report that definitive evidence for DNA binding of acrylonitrile was not obtained but that there was some evidence for oxidative DNA damage in the brain but not Zymbal’s glands. Potential mechanisms of action for acrylonitrile carcinogenicity are discussed.

The outcomes of other in vitro genotoxicity studies of acrylonitrile were mixed.9−11,14 It produced chromosome aberrations in Chinese hamster ovary cells and liver and lung fibroblasts, but no chromosome damage was reported in rat hepatocytes or in the micronucleus assay on human bronchial epithelium cells.9 A sister chromatid exchange assay was positive in Chinese hamster ovary cells but not in hepatocytes and human lymphocytes.9 The results of unscheduled DNA synthesis measurement in cultured mammalian cells were inconclusive.11 While some studies showed the direct reaction of acrylonitrile with DNA,13,20 others found no binding of [1-14C]-acrylonitrile incubated with DNA.15 A study using cultured F344 rat hepatocytes21 demonstrated that acrylonitrile produced dose-dependent cytotoxicity while depleting reduced glutathione (GSH), a peptide involved in detoxication of acrylonitrile in rodents and humans.22,23 Also, a very small fraction of acrylonitrile bound to DNA. The authors of the study concluded that acrylonitrile binds to GSH and proteins as a result of direct reaction rather than through its metabolite.21 Other studies demonstrate that both acrylonitrile and CEO react with GSH.15,24 While mice have higher rates of acrylonitrile oxidation to CEO compared to those of rats and humans,9,25 their capacity for GSH conjugation of acrylonitrile and CEO is much greater than that in rats.25,26 Binding of acrylonitrile to proteins has also been documented in vivo in several rat tissues, including the liver, lung, and spleen.27−30 The results of in vivo studies of acrylonitrile genotoxic potential also were mixed.9,10,14 Acrylonitrile produced DNA strand breaks detected by the comet assay in the livers of SD rats but not in the glandular stomach.31 No chromosomal aberrations were reported in bone marrow of mice and rats exposed to acrylonitrile. The mouse micronucleus assay and the dominant-lethal assay in rats and mice were also negative.9,11,32,33 Positive unscheduled DNA synthesis results in rats with acrylonitrile were obtained only at a near lethal dose.34 The negative outcomes in vivo possibly result from rapid GSH conjugation of acrylonitrile and CEO and consequent detoxication.9,24 Importantly, the target tissues of acrylonitrile carcinogenicity are those with low biotransformation capability. Thus, Guengerich et al.15 concluded that it is possible that in rats, the brain is a target organ of acrylonitrile due to the low ability for its detoxication through conjugation with GSH. Pilon and colleagues23 also found increased association of 2,3-14Cacrylonitrile with DNA only in target organs in GSH-depleted F344 rats. While in some studies in humans an association was found between the polymorphism of GSH transferases and CYP 450 2E1 and toxicity of acrylonitrile,35 in other studies no relationship between GSH transferases polymorphism and acrylonitrile-derived hemoglobin adducts was found.36 Some in vivo studies using 2,3-14C-acrylonitrile yielded findings suggesting that it binds to DNA in several organs of rats, including the brain, kidney, lung, liver, stomach, and testes,37−42 not all of which are target organs. Moreover, clear evidence of DNA adduct formation after exposure to acrylonitrile, in particular in the primary target organ, the brain, has not been obtained. For example, Farooqui and Ahmed40 detected higher levels of DNA binding in the brain compared to the liver of rats after oral administration of 2,3-14C-acrylonitrile. However, according to the authors, the results are more consistent with metabolic incorporation of the radiolabel from 14C-acrylonitrile into normal purine 2′-

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MATERIALS AND METHODS

Materials. Acrylonitrile (CAS: 107-13-1; >99% pure as reported by supplier) was purchased from Sigma-Aldrich (St. Louis, MO, USA; lot # 29296PMV). The 2,3-14C-acrylonitrile (CAS: 107-13-1; 97.5%; lot number 120412; specific activity 3.3 mCi/mmol), supplied as a neat liquid stabilized with 40 ppm of polymerization inhibitor, hydroquinone, in a sealed ampule, by American Radiolabeled Chemicals, Inc. (ARC, St. Louis, MO, USA). The positive control for DNA binding and DNA adduct formation, 7-14C-benzo[a]pyrene (CAS: 93127-18-5; >98.6%; lot number 120319; specific activity 26.6 mCi/ mmol), was obtained from American Radiolabeled Chemicals, Inc. (ARC, St. Louis, MO, USA). For acrylonitrile, drinking water was used as a vehicle. A 0.5% solution of methyl cellulose (CAS: 9004-67-5) supplied by ICN Biomedicals Inc. (Aurora, OH, USA; lot # 1489B) in drinking water (400CPS for 2% aqueous solution) was used as a vehicle for benzo[a]pyrene at the dose 5 mg/kg bw. The positive 1471


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Chemical Research in Toxicology Table 1. Experimental Designa

group ID

time-point of termination (h)

dosing details

F344 control SD control

drinking water daily for 28 days (negative control)

3

F344 AN SD AN F344 DW + 14C-AN SD DW + 14C-AN

acrylonitrile at 100 ppm in drinking waterc daily for 28 days

3

drinking water daily for 27 days, on day 28 a single oral gavage dose of 14Cacrylonitrile 100 μCi (3.7 MBq) at 11 mg/kg bw

3

F344 AN + 14C-AN SD AN + 14C-AN

acrylonitrile at 100 ppm in drinking waterc daily for 27 days, on day 28 a single oral gavage dose of 14C-acrylonitrile 100 μCi (3.7 MBq) at 11 mg/kg bw

3

F344 DW + 14C-BP SD DW + 14C-BP

drinking water daily for 26 days, on day 27 14C-benzo[a]pyrene given as a single oral gavage dose of 8 μCi at 5 mg/kg bw (positive control)

24

DNA binding analysis in brain

cometd analysis in the brain and Zymbal’s gland

number of ratsb/strain

number of ratsb/strain

6/F344 6/SD

3/F344 3/SD 3/SD plus HP 3/F344 3/SD

3/F344 3/SD 3/F344 3/SD 3/F344 3/SD

a14

C-AN, 2, 3-14C-acrylonitrile; 14C-BP, 7-14C-benzo[a]pyrene in 0.5% methylcellulose; AN, acrylonitrile dissolved in drinking water; DW, drinking water; FPG, E. coli formidopyrimidine-DNA glycosylase enzyme digestion assay, 0.5U/slide; hOGG1, human 8-hydroxyguanine-DNA glycosylase enzyme digestion assay, 0.3U/slide; h, hours after the last dose; HP, hydrogen peroxide, exposure to slides at 30 μmol/slide, positive control for FPG and hOGG1 assays. bOnly female F344 (Fischer 344) and SD (Sprague−Dawley) rats were utilized. cPrepared fresh at least twice a week. d Conventional alkaline single cell gel electrophoresis (comet), FPG, and hOGG1 enhanced comet assays. kg28,37 or 33 μCi/kg,60 or even 600 μCi per rat16 have shown that 100 μCi (3.7 MBq) would be appropriate. Benzo[a]pyrene forms DNA adducts in several rat tissues, including the brain and liver,57 which have been well characterized and detected with high sensitivity by 32Ppostlabeling.58 Accordingly, 14C benzo[a]pyrene (8 μCi) at 5 mg/kg bw was used as a positive comparator. The control group received drinking water ad libitum for 28 days. In test groups used to assess DNA binding, rats received 100 ppm nonradiolabeled acrylonitrile in the drinking water, prepared fresh at least twice a week, for 27 days, to allow for possible induction of biotransformation systems and accumulation of DNA damage, and, on the 28th day, a dose of 100 μCi (3.7 MBq) of 14C-acrylonitrile was administered by a single gavage at 11 mg/kg bw in water. The comparison group received drinking water for 27 days, and, on the 28th day, a dose of 100 μCi (3.7 MBq) of 14C-acrylonitrile was administered by a single gavage at 11 mg/kg bw in water. Rats were subsequently terminated 3 h later. In 14C benzo[a]pyrene group, rats were given drinking water for 26 days, and on the 27th day, 8 μCi of 14 C benzo[a]pyrene at 5 mg/kg bw was administered by a single gavage dose to 3 rats of each strain. Rats were terminated 24 h later. In groups utilized for conventional and enhanced comet assays, unlabeled acrylonitrile was administered at 100 ppm in the drinking water ad libitum for 28 days. Rats were observed twice daily. Body weights were obtained on day 1 before dosing and weekly thereafter and before euthanasia or prior to gavage of 14C-acrylonitrile or 14C-benzo[a]pyrene. At termination, euthanasia was achieved by cardiac puncture under CO2 anesthesia. Groups dosed with acrylonitrile were terminated 3 h after the gavage in order to avoid repair of adducts and thereby capture the maximum adducts present. Rats dosed with 14C benzo[a]pyrene were terminated 24 h after dosing, based on the protocol used in the study that investigated benzo[a]pyrene DNA adducts formation using the NPL assay58 and data on the persistence of benzo[a]pyrene DNA adducts.57 Immediately after termination, the brain and Zymbal’s gland samples from appropriate groups were rinsed in ice cold saline and added to individual vials for analysis in the conventional and enhanced comet assay. Individual Zymbal’s gland tissue was insufficient to allow isolation of sufficient quantities of DNA for DNA binding analysis. Therefore, pooled glands from both sides, i.e., 4 to 6 pieces bilaterally, were used for conventional and enhanced comet assays. Measurement of AN-DNA Binding. Binding of 14C-acrylonitrile to rigorously purified DNA from rat brain (target) tissue was measured

control for the enhanced comet assay, hydrogen peroxide, was obtained from Aldrich Chemical Co., Inc. (St. Louis, MO, USA; lot # 19414BI); the dose used was 30 μmol. Test System. The experimental design is given in Table 1. Two strains of female rats, i.e., Spartan SD and F344, were used in this study as these were the strains used in the six carcinogenicity studies.1−4 Females were used because in all chronic studies, the daily acrylonitrile in drinking water exposure (in mg/kg bw/d) was higher than that in males and also because the incidence of brain tumors in some studies was numerically greater compared to that in males.2−4 The rats were 6 weeks of age at the start of the study and were housed in the Department of Comparative Medicine of New York Medical College, an Association for Assessment and Accreditation of Laboratory Animal Care certified facility under the supervision of a veterinarian. Control, acrylonitrile, and 14C-benzo[a]pyrene dosed groups each were housed in separate rooms to prevent any potential cross-contamination of test materials. The rats were obtained from Charles River Laboratories International, Inc. (Wilmington, MA, USA) and housed 3 to 4 per cage in solid bottomed polycarbonate cages with irradiated corn cob bedding. The room temperature was maintained at 72 ± 10 °F and relative humidity at 55% ± 20. Adequate fresh air was supplied to the animal rooms. Twelve hours of continuous low level fluorescent lighting (5 ft candles) was provided daily followed by 12 h of darkness. Water was available ad libitum throughout the study and was supplied in plastic drinking bottles together with acrylonitrile at 100 ppm. Fresh solutions were prepared at least twice a week. Monitoring of the drinking water for microbiological and chemical contaminants was routinely conducted. The NIH-07 (supplied by Purina as 5018 NIH meal) diet was provided ad libitum throughout the study. Monitoring of diet for nutritional components and chemical contaminants was provided by the suppliers. Upon receipt, the rats were maintained under observation for a minimum of 1 week. Body weights of animals selected for inclusion in the study did not vary by more than 20% from the mean group body weight. Identification was done by indelible tail marks and recorded in the raw data. All doses were selected on the basis of previous studies. Radiochemicals were administered by gavage for precise dosing. On the basis of findings with a known genotoxic rat carcinogen (tamoxifen), for which compelling findings of DNA binding using AMS were obtained with the administration of 5.4 μCi (0.2 MBq),59 administered dose of 14C-acrylonitrile was chosen to be 100 μCi (3.7 MBq) per rat. Earlier LSC studies with 14C-acrylonitrile using 50 μCi/ 1472


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Figure 1. Weekly body weight gain in F344 and SD female rats from control groups and groups that received acrylonitrile (AN) at 100 ppm in drinking water daily for 28 days. water followed by a single gavage dose of 14C-acrylonitrile, and F344 and SD rats dosed with 14C-benzo[a]pyrene, and were enzymatically digested into 2′-deoxyribonucleoside 3′-monophosphates using micrococcal nuclease and spleen phosphodiesterase groups. The digest was then enriched for DNA modified bases using nuclease P1 digestion,71 which provides for better sensitivity, except for DNA adducts that are sensitive to this treatment. DNA bases were then labeled using 100 μCi γ-32P adenosine triphosphate (∼6000 Ci/mmole, PerkinElmer, Waltham, MA, USA). The labeled modified bases were then resolved using two- or three-directional thin-layer chromatography (TLC) systems. Suitable solvent systems for the development of the TLC plates were established for a variety of adducts.70,72−75 D1 direction solvent; 2.3 M sodium phosphate, pH 5.5. D3 direction solvent; 3.3 M lithium formate/8.0 M urea pH 3.5. D4 direction solvent; 1 M Lithium chloride/0.425 M Tris HCl/6.8 M urea, pH 8.0. The radioactivity on the TLC plates was detected using a Molecular Dynamics Storm 860 system with exposure times of 14 h (GE Health Care Life Sciences, Edison, NJ, USA). DNA adducts in 108 of normal nucleotides were ascertained and calculated. Alkaline Single Cell Gel Electrophoresis (Comet) Assay. The conventional52−54 and enhanced55 alkaline comet analyses were performed on fresh cells from the brain and Zymbal’s gland samples of F344 and SD rats from the vehicle control group and groups that received acrylonitrile at 100 ppm in drinking water for 28 days. Experimental design is provided in Table 1. The conventional comet assay was enhanced with E. coli formamidopyrimidine-DNA glycosylase (FPG) enzyme digestion to assess general oxidative damage 55,76,77 and human 8-hydroxyguanine-DNA glycosylase (hOGG1) enzyme digestion to assess specifically the formation of 8oxodG.78,79 The hOGG1 cloned gene was carried by an E. coli strain.80 As a positive comparator for FPG and hOGG1 assays, one set of slides was treated with a positive comparator hydrogen peroxide as detailed below. The brains and Zymbal’s glands were rinsed and then placed in cold Hank’s balance salt solution (HBSS, Gibco, Grand Island, NY, USA) on ice. To prepare cells for comet analysis, a small piece of tissue (about 250 mg) was placed in 1 mL of cold HBSS containing 20 mM EDTA and 10% DMSO and minced into fine pieces, and the pieces were allowed to settle. Then, aliquots of the cell suspensions were used. The comet assay was performed according to Tice et al.52 The protocol previously described in detail74 was used. DNA migration was analyzed by fluorescence microscopy using a Nikon OPTIPHOT microscope. The percentage of DNA-in-tail was determined using the Comet Score software v 1.5 (TriTek Corp, Sumerduck, Virginia), counting >150 cells per sample. Median % tail DNA for each slide was

by LSC and AMS, the latter being orders of magnitude more sensitive than LSC,49 the conventional method, which has been used to date. In pilot experiments, the DNA isolated from the brain using DNAzol61 contained material which redissolved poorly. To improve isolation, DNA was purified either by a one or three step purification processes. In one step purification, DNA was purified using Qiagen G100 columns62 following protease and RNase treatment of the brain tissue. This allows separation on the columns of proteins, polysaccharides, and low molecular weight compounds which elute first, followed by oligonucleotides, and finally DNA with increasing salt concentration. Three step purification consisted of one step purification by Qiagen columns, followed by the purification process consisting of DNAzol, and finally repurification using digestion with proteinase K and chloroform/iso-amyl alcohol extraction and ethanol precipitation.63 The purity of DNA was measured using a Cary 1E UV/visible spectrophotometer (Varian Optical Spectroscopy Instruments, Mulgrave, Victoria, Australia). The extensively purified DNA samples (260/280 and 260/230 nm absorbance ratios greater than 1.8 and 2.0, respectively) were assessed for associated radioactivity by LSC counting of each sample (DNA 5 μg) for 10 min and up to 10 cycles to yield average values. For AMS analysis, vacuum-dried DNA samples were shipped on dry ice to the DirectAMS facility (Bothell, WA, USA) a Radiocarbon Dating Service, and subsidiary of Accium Biosciences (Seattle, WA, USA), where it was converted, via carbon dioxide, for analysis by standard AMS methodology49,64−66 and equipment as described by Accium.67 The values reported herein were based on a 5 μg of DNA sample being counted. Agarose Gel Electrophoresis. Agarose gel preparation and electrophoresis were conducted following the guidelines described by Sambrook et al.68 Serial dilutions (0.11, 0.22, and 1.10 μg) of genomic DNA from the brain of F344 female rats dosed with 14C-acrylonitrile were mixed 1:4 with loading buffer (0.5 μg/mL ethidium bromide in 1× Tris-borate EDTA (TBE) buffer, pH 8.0) and 10 μL aliquots were transferred into a 0.7% agarose gel prepared using 1× Tris-acetate EDTA (TAE) buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA). A 10 kb MassRuler DNA Ladder (MBI Fermentas, Amherst, NY, USA) was used as molecular weight marker. Electrophoresis was then conducted at 100 V for 2.5 h. Several concentrations (0.17, 0.34, and 1.70 μg) of calf thymus DNA (Sigma Chemical Co., St Louis, MO, USA) served as a control. Nucleotide 32P-Postlabeling (NPL) Assay for DNA Adducts. DNA from brain was processed for adducts analysis by NPL method of Randerath,51 as previously described.50,69,70 DNA samples (10 μg) were taken each from the vehicle control group, groups of F344 and SD rats dosed with acrylonitrile in drinking 1473


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Table 2. Calculated 14C Binding in DNA Enriched Samples Isolated from the Brains of Rats Dosed with a Single Oral Gavage 14C-Acrylonitrile or 14C-Benzo[a]pyrene Dose in the Accelerator Mass Spectrometry (AMS) Assaya

determined, and the mean values was calculated for each sample; the group mean was calculated as an average of individual sample means. Statistical Analysis. Statistical analysis was performed using SigmaStat for Windows, version 3.11.0 (Systat Software Inc., Chicago, IL, USA), with the most appropriate statistical analysis for the data sets, which included the Holm−Sidak pairwise multiple comparison procedures, the Kruskal−Walis one-way analysis of variance (ANOVA) on ranks, and multiple comparisons.

ID

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F344 control

RESULTS No deviations occurred in ambient temperature, relative humidity, or any other condition in the animal rooms that would affect animal health or the integrity of the study. No compound-related deaths or clinical findings occurred in the study. Both strains gained weight throughout the study period with no effect of acrylonitrile dosing (Figure 1). DNA Binding. Isolated brain DNA samples were assessed for associated radioactivity by the conventional LSC method, through either a one or a three step purification process described in the Materials and Methods section. Only brain samples were used since the Zymbal’s glands were too small to yield sufficient quantities of DNA. In brain samples subjected to either purification process, decay counting for 10 cycles of 10 min each revealed no significant difference in the levels of radioactivity in groups dosed with 14C-acrylonitrile or 14Cbenzo[a]pyrene over background (results not shown). In the AMS analysis, no radioactivity was associated with control rat brain samples. Groups of F344 or SD rats dosed with 14C-benzo[a]pyrene had a level of associated radioactivity in the brain (Table 2 and Figure 2) as expected based on the level of DNA modification described in the literature.57 Groups that received acrylonitrile for 28 days (27 days of nonradiolabeled acrylonitrile in drinking water and single dose of 14 C-acrylonitrile on day 28 via gavage) and those that received only a single gavage dose of 14C-acrylonitrile on day 28 exhibited similar levels of radioactivity in the brain tissues of the rats from both strains (Table 2 and Figure 2). Thus, dosing with acrylonitrile for 27 days in drinking water did not affect levels of associated radioactivity. Levels were numerically greater in F344 rat samples but not significantly different from those in SD rats (Table 2). The associated radioactivity, if due to DNA-binding, is calculated to amount to about 7−13 adducts per 108 of normal nucleotides (Table 2). In order to explore the nature of the 14C-association with brain DNA from F344 rats which received acrylonitrile in drinking water for 27 days followed by a single oral gavage of 14 C-acrylonitrile, electrophoretic separation of high-molecular weight molecules using agarose gel was conducted (Figure 3). The genomic DNA prepared from the rat brain was less fragmented compared to that of commercially obtained calf thymus DNA. The rat brain samples were mostly composed of DNA but had some high molecular weight material that did not enter the gel. This could indicate the presence of either crosslinked DNA or residual proteins which were not digested by proteinase K. Adduct Formation. In the NPL analysis of 28 day brain samples (14C-acrylonitrile- or 14C-benzo[a]pyrene-dosed), using nuclease P1 enrichment, in rats administered benzo[a]pyrene, a single DNA adduct was detected in F344 rat brain DNA reaching a level of 0.015 in 108 of normal nucleotides, while the SD rat brain DNA adduct reached a level of 0.01 in 108 of normal nucleotides. Acrylonitrile-dosed F344 and SD rat brain DNA yielded no detectable adduct (Figure 4A and B).

SD control

F344 DW + 14CAN

SD DW + 14 C-AN

F344 AN + 14CAN

SD AN + 14 C-AN

F344 DW + 14CBP

SD DW + 14 C-BP

dpm/gC

error

net dpm/ gC

2.793

0.039

0.02

0

0±0

2.802 2.731 2.842

0.034 0.029 0.048

0.02 −0.05 0.06

0 0 0.73

0.03 ± 0.07

2.732 2.835 14.257

0.028 0.04 0.076

−0.04 0.06 11.48

0.5 0.65 128.56

13.04 ± 4.03

10.91 18.107 8.795

0.17 0.097 0.098

8.13 15.33 6.02

91.07 171.67 67.39

7.11 ± 2.11

7.435 11.16 18.7

0.078 0.1 0.13

4.66 8.38 15.92

52.16 93.87 178.31

13.2 ± 4.63

10.436 14.568 9.312

0.071 0.101 0.07

7.66 11.79 6.54

85.77 132.04 73.18

7.38 ± 1.5

8.058 10.742 2.962

0.055 0.066 0.037

5.28 7.97 0.19

59.14 89.19 3.36

0.95 ± 0.55

3.036 2.983 3.011

0.035 0.036 0.036

0.26 0.21 0.23

13.92 11.07 4.25

1.22 ± 0.79

3.007 3.151

0.042 0.032

0.23 0.37

12.36 20.1

nmole AN/mol DNA based on 5 μg

adduct in 108 nts mean value ± SD

a14

C-AN, 2, 3-14C-acrylonitrile; 14C-BP, 7-14C-benzo[a]pyrene in 0.5% methylcellulose; AN, acrylonitrile; AMS was calculated in dpm/μg sample; C, carbon; DW, drinking water; nts, nucleotides.

DNA Breakage. The conventional comet assay results on isolated cells were negative for all groups from both the brain and Zymbal’s glands of both strains of rats dosed with acrylonitrile at 100 ppm in drinking water for 28 days, as well as for positive control samples treated with hydrogen peroxide (Table 3 and Figure 5A and B). The enhanced comet assay results showed DNA breakage with hydrogen peroxide in both brain and Zymbal’s gland cells digested with FPG or hOGG1 prior to electrophoresis (Figures 5A and 5B). Dosing of F344 and SD rats with acrylonitrile produced a significant increase in the percentage of tail DNA (2.1- and 2.6-fold increase, respectively) in brain samples when the cells were digested with FPG but not with the hOGG1 enzyme (Table 3 and Figure 5A). In contrast, the Zymbal’s gland cell comet assay, with both FPG and hOGG1 enzymes digestion, was negative (Figure 5B).

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DISCUSSION The potential of acrylonitrile to produce DNA damage in two rat primary target tissues of carcinogenicity, the brain and 1474


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Figure 2. Calculated 14C binding assessed by accelerator mass spectrometry (AMS) in brain DNA enriched samples isolated from F344 and SD female rats dosed with 14C-acrylonitrile (AN) or 14Cbenzo[a]pyrene (BP). Accelerator mass spectrometry calculated in dpm/μg of DNA as binding values (moles of AN or BP per 108 of normal nucleotides). DW, drinking water.

Figure 4. Chromatograms (A) and total DNA adducts levels (B) in the brain samples from Fischer 344 (F344) and Sprague−Dawley (SD) female rats from control groups and groups dosed with 14Cacrylonitrile (AN) or 14C-benzo[a]pyrene (BP) detected with the nucleotide 32P-postlabeling (NPL) assay. Adducts were resolved in the second and third directions of chromatography and are indicated by arrows. Obtained with nuclease P1 enrichment method. DW, drinking water.

water and a single dose of 14C-acrylonitrile on day 28 by gavage) to provide for prolonged exposure and any modulation of biotransformation. No difference in the association of 14C of acrylonitrile with DNA was observed between groups that received 1-day (27 days of drinking water and a single dose of 14 C-acrylonitrile on day 28 via gavage) and 28-days acrylonitrile dosing, and hence, no evidence was obtained for either induction or inhibition of biotransformation processes with extended dosing. Association of 14C-acrylonitrile with DNA was assessed by LSC and AMS. Only brain samples were used for analysis since the Zymbal’s glands were too small to allow isolation of sufficient quantities of DNA. No 14C association in the acrylonitrile samples was detectable by LSC. Using extensively purified DNA in AMS analysis, which is orders of magnitude more sensitive than LSC,49 association of 14C of acrylonitrile in the rat brain was detected (Figure 2). The positive control

Figure 3. Electrophoretic separation of high-molecular weight material using agarose gel. Br, brain DNA from Fischer 344 female rats dosed with 14C-acrylonitrile. CT, calf thymus DNA. Most material migrated with the DNA ladder, but some large molecular weight material did not enter the gel.

Zymbal’s glands, was studied by several complementary methodologies. The study design involved 28 day acrylonitrile dosing (27 days of nonradiolabeled acrylonitrile in drinking 1475


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Chemical Research in Toxicology Table 3. Calculated Percentage of DNA in the Tail in the Brain and Zymbal’s Gland of Control Rats and Rats Dosed with Acrylonitrile in Drinking Water for 28 Days Using Alkalinea, FPG, and Hogg1comet Analysisb brain group ID HP

F344 control

SD control

F344 AN

SD AN

Zymbal’s gland

type of assay

mean ± SD

mean ± SD

alkaline FPG hOGG1 alkaline FPG hOGG1 alkaline FPG hOGG1 alkaline FPG hOGG1 alkaline FPG hOGG1

8.84 ± 5.61 25.46 ± 2.96* 23.03 ± 5.14* 9.74 ± 5.82 9.41 ± 2.74 10.25 ± 2.84 7.54 ± 2.19 8.71 ± 3.63 8.17 ± 4.35 8.84 ± 4.75 19.95 ± 4.24* 8.23 ± 5.35 10.41 ± 2.36 22.52 ± 3.5* 11.11 ± 2.75

12.38 ± 4.46 29.56 ± 6.22* 29.4 ± 4.5* 10.04 ± 6.57 10.39 ± 4.23 11.22 ± 8.53 9.53 ± 6.2 11.05 ± 3.55 13.36 ± 5.46 8.94 ± 4.04 12.44 ± 5.37 9.15 ± 4.7 10.08 ± 7.4 9.9 ± 3.27 12.62 ± 6.43

a

Conventional alkaline single cell gel electrophoresis (comet) assay. AN, acrylonitrile dissolved in drinking water; DW, drinking water; FPG, E. coli formidopyrimidine-DNA glycosylase enzyme digestion assay, 0.5U/slide; hOGG1, human 8-hydroxyguanine-DNA glycosylase enzyme digestion assay, 0.3U/slide; HP, hydrogen peroxide, exposure to slides at 30 μmol/slide, positive control for FPG and hOGG1 assays; * denotes significant (p < 0.05) difference from the control group.

b

Figure 5. Conventional (alkaline) and enhanced comet assay results from brains (A) and Zymbal’s glands (B) of F344 and SD female rats dosed with AN in drinking water. In the enhanced comet assay, digestion of samples either with E. coli formamidopyrimidine-DNA glycosylase (FPG) or human 8-hydroxyguanine-DNA glycosylase (hOGG1) enzymes was performed. Hydrogen peroxide (HP) was used as a positive control. (*, denotes significant (p < 0.05) difference from the control group).

(14C-benzo[a]pyrene) was close to the expected values based on the levels of adducts reported by Stowers and Anderson.57 The acrylonitrile radioactivity associated with the DNA in the current study, however, cannot be conclusively attributed to DNA adduct formation. Despite extensive purification of the DNA, there could still be some associated proteins, as evident from agarose gel electrophoresis of the genomic DNA from the brain (Figure 3). Owing to the greater reactivity with the soft nucleophilic sulfur centers in proteins compared to the harder nitrogen sites found in DNA,81 binding to protein could be substantial. Others have reported that purified DNA samples contain low levels (occasionally up to 10%) of contaminating protein.82 There is also the possibility of metabolic incorporation of metabolized acrylonitrile, i.e., 14C utilized in the synthesis of normal nucleotides of DNA. In experiments by Pilon et al.,23 in which 4 mg of 14Cacrylonitrile was administered by gavage to male F344 rats, binding of the compound to DNA in the brain of rats was not detected in dialyzed samples assessed by LSC, while total radioactivity in the brain tissue was the lowest compared to that of other tissues analyzed in the study. High doses of 14Cacrylonitrile (46.5 mg/kg bw, LD50) resulted in very high binding levels of approximately 57 μmol acrylonitrile bound/ mol DNA (40 in 106 nucleotides) in the brain of male SD rats detected by LSC, 40 although this may reflect protein contamination. Studies by Solomon et al.13 and Yates et al.18 described several DNA adducts produced by CEO, a reactive metabolite of acrylonitrile, using ultraviolet spectroscopy, NMR spectroscopy, and mass spectrometry. In the present study, using AMS methodology, DNA-associated 14C-acrylonitrile radioactivity in the brains of rats of both strains was detected (Table 2 and Figure 3). The associated radioactivity is

calculated to represent less than 15 adducts per 108 nucleotides (Table 2), if it is in fact DNA binding. The biological significance of such low level of binding is not known, due to the presence of protection mechanisms against genotoxicity, such as binding to noncoding regions of DNA and DNA damage repair.83−85 DNA binding was not confirmed by the other methods applied, i.e., NPL and the comet assay. In the NPL assay for adducts, samples from rats administered a single dose of benzo[a]pyrene displayed adducts as expected based on the previously published evidence of benzo[a]pyrene binding to brain DNA,58 whereas acrylonitrile samples, even with 28 days of dosing, did not (Figure 4A and B). An adduct formed by CEO, e.g., cyano-hydroxyethyl deoxythymidine, would have a molecular weight of 311 g/mol18 and might be detectible, although the sensitivity of the method for detection of potential acrylonitrile adducts is not known since no standard was used. Nevertheless, the negative outcome provides no support for the interpretation that 14C-acrylonitrile association with brain DNA represents DNA binding. Dosing with acrylonitrile in both strains produced negative conventional comet assay results in both the brain and Zymbal’s glands, which is consistent with an absence of DNA binding in brain. When the brain samples were treated with oxidative DNA damage repair enzymes, prior to comet 1476


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previously shown to be induced by exposure to various chemicals.90,91 Further elucidation of the role of protein binding of acrylonitrile seems warranted. In summary, not having identified direct DNA damage by acrylonitrile in two target tissues, the brain and Zymbal’s gland in F344 and SD rats, we conclude that definitive evidence of a role for DNA binding in acrylonitrile carcinogenicity was not obtained. In contrast, some evidence of oxidative damage produced by acrylonitrile in brain tissues of both rat strains was present, as evidenced by positive results of enhanced comet assay with FPG enzyme digestion. Also, acrylonitrile binding to protein could contribute to its carcinogenicity. Thus, acrylonitrile carcinogenicity may be multifactorial and could involve several physiologic perturbations.

measurement, positive results were obtained with FPG but not with hOGG1 (Table 3 and Figure 5A). The DNA FPG digestion removes a variety of oxidized bases,55,76 as well as imidazole ring-opened purines either modified at N7 or C8 positions in DNA.86,87 FPG digestion yielded positive results after hydrogen peroxide treatment (Figure 5A and B). Thus, the findings with FPG are suggestive of the presence of DNA oxidative adducts but not specifically 8-oxodG since hOGG1 digested samples were negative. The current findings are consistent with previous positive findings of oxidative DNA damage induced by acrylonitrile detected by electrochemical detection43 and the FPG digestion enhanced comet assay44−46 that have been reported in vitro and in vivo, including the DI TNC1 rat astrocyte cell line and brain tissue of SD rats.45,46 The Zymbal’s glands, also a target tissue for acrylonitrile carcinogenicity, were negative in both the conventional comet and also the enzyme enhanced comet assays (Figure 5B), suggesting a complete lack of DNA breaks in both strains of rats. Overall, under the test conditions definitive evidence of DNA binding of acrylonitrile was not found in two target tissues of acrylonitrile carcinogenicity in two strains of rats with several methodologies. Accordingly, other mechanisms, i.e., oxidative stress, could be involved in the induction of neoplasms. There are several possible indirect genotoxic or epigenetic mechanisms which could be the basis for acrylonitrile carcinogenicity. Among them, acrylonitrile-induced oxidative stress has been extensively studied. Several studies report acrylonitrile mediated increases in production of oxidative lesions (8-oxodG) and increase in the formation of hydroxyl radicals in astrocytes of rats and humans in vitro and in rat brain in vivo.43−46,88,89 Depletion of GSH by acrylonitrile was shown to play a role in the exacerbation of the oxidative damage45 and to facilitate the protein binding of acrylonitrile.28 This peptide is involved in the detoxication of acrylonitrile, and its depletion would result in the higher levels of acrylonitrile in the cell.21 In a nontarget rat tissue, the liver, the formation of oxidative lesions was described in vivo,43 although at levels lower than that in the brain. In the present study, evidence of oxidative DNA damage was found in the brain but not in the Zymbal’s glands of rats of both strains. Accordingly, oxidative damage may contribute to acrylonitrile carcinogenicity but does not appear to be a sufficient or necessary mechanism of action. Importantly, in rats, acrylonitrile has been shown to selectively bind to the Cys 186 residue in carbonic anhydrase III, protein in the liver, which plays an important role as an intracellular antioxidant.29 Such binding can impair the function of carbonic anhydrase III, which contributes to oxidative damage. Caito and colleagues47 investigated the inflammatory response in the microglia and astrocytes in vitro after exposure to acrylonitrile. They concluded that the oxidative damage produced by acrylonitrile initiates an inflammatory cascade with release of pro-inflammatory mediators in the microglia by the brain cells, with astrocytes reacting to the exposure first. The inflammation and proliferation of microglia could also be an important factor in the development of brain tumors after acrylonitrile exposure.7 Irreversible binding of acrylonitrile to proteins has been previously shown;15,27−30 however, the consequences of such binding are yet not well described. It is plausible that exposure to acrylonitrile can modify histone proteins. Such alterations play an important role in carcinogenesis and have been

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AUTHOR INFORMATION

Corresponding Author

*Phone: 914-594-3085. Fax: 914-594-4163. E-mail: Gary_ [email protected]. ORCID

Gary M. Williams: 0000-0002-5159-8124 Funding

This study was supported by funding from Global Acrylonitrile Product Stewardship. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Sharon Brana for preparation of the manuscript. DEDICATION This article is dedicated to the memory of our colleague, Dr. Alan Jeffrey, now deceased, who made a major contribution to this project.

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ABBREVIATIONS AMS, accelerator mass spectrometry; CEO, 2-cyanoethylene oxide; comet, alkaline single cell gel electrophoresis assay; F344, Fischer 344 strain of rat; FPG, E. coli formamidopyrimidine-DNA glycosylase enzyme; GSH, reduced glutathione; hOGG1, human 8-hydroxyguanine-DNA glycosylase enzyme; LSC, liquid scintillation counting; NPL, nucleotide 32Ppostlabeling assay; 8-oxodG, 8-oxo-deoxyguanine; SD, Sprague−Dawley strain of rat; TLC, thin layer chromatography

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REFERENCES

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