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Chemosphere 67 (2007) 1164–1170

N-Nitroso compounds produced in deer mouse (Peromyscusmaniculatus) GI tracts following hexahydro-1,3,5-

trinitro-1,3,5-triazine (RDX) exposure

Xiaoping Pan a,*, Baohong Zhang a, Jordan N. Smith a, Michael San Francisco b,Todd A. Anderson a, George P. Cobb a

a The Institute of Environmental and Human Health, Department of Environmental Toxicology, Texas Tech University,

Lubbock, TX 79409-1163, USAb Department of Biological Sciences, Texas Tech University, Lubbock, TX 79401, USA

Received 24 June 2006; received in revised form 29 October 2006; accepted 31 October 2006Available online 16 January 2007

Abstract

Given the potent carcinogenic effects of most N-nitroso compounds, the reductive transformation of the common explosive hexahy-dro-1,3,5-trinitro-1,3,5-triazine (RDX) to a group of N-nitroso derivatives, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexa-hydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) in the environment have causedconcerns among the general public. Questions are arising about whether the same transformations also occur in mammals, and if true,to what extent. This study investigated the N-nitroso derivatives production in the deer mouse GI tract following RDX administration.Findings verified that such transformations do occur in the mammalian GI tract at notable levels: the average MNX concentrations indeer mice stomach were 85 lg/kg and 1318 lg/kg for exposure to 10 mg/kg and 100 mg/kg diet, respectively. DNX in stomach were217 lg/kg for the 10 mg/kg dose group and 498 lg/kg for the 100 mg/kg dose group. Changes in other toxic endpoints including bodyweight gain, food consumption, organ weight, and behavior were also reported.� 2006 Elsevier Ltd. All rights reserved.

Keywords: RDX; MNX; DNX; TNX; N-nitroso compounds (NOCs); GI tract; Deer mice; Body weight; Organ weight; Behavior

1. Introduction

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is animportant explosive (ATSDR, 1995). As many as 583 mili-tary sites in the U.S. have been identified as contaminatedwith RDX, with an additional 88 sites suspected of havingRDX contamination (Walsh and Jenkins, 1992). The USEnvironmental Protection Agency has classified RDX as apotential human carcinogen (Class C) (EPA, 1988). Thisclassification is primarily based on elevated tumor incidencein male and female B6C3F1 mice; a statistically significantincrease in hepatocellular carcinomas and adenomas (com-

0045-6535/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.chemosphere.2006.10.077

* Corresponding author. Tel.: +1 806 885 4567.E-mail address: xiaoping.pan@tiehh.ttu.edu (X. Pan).

bined) was observed in females receiving as little as 7.0 mg/kg/day RDX orally for 24 months (Lish et al., 1984). How-ever, the carcinogenic mechanism of RDX remains unpro-ven. Since McCormick et al. first proposed that someanaerobic bacteria can sequentially reduce RDX to a groupof N-nitroso derivatives, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) (McCormick et al., 1981) (Fig. 1), manyadditional studies have observed this group of N-nitrosoderivatives in anaerobic sludge, anaerobic reactors, andother samples (Kitts et al., 1994b; Young et al., 1997a;Young et al., 1997b; Hawari et al., 2000a; Adrian andArnett, 2004; Bhushan et al., 2004). More importantly, theseN-nitroso derivatives have been detected in groundwater

Fig. 1. Chemical structures of RDX, MNX, DNX, TNX, N-nitrosopiperidine, and N,N 0-dinitrosopiperazine.

X. Pan et al. / Chemosphere 67 (2007) 1164–1170 1165

samples near an RDX-contaminated site (Beller and Tieme-ier, 2002), suggesting that such reductive transformationof RDX does occur in the natural environment. Despiteample studies revealing this type of RDX transformationin the environment, the in vivo RDX transformation to itsN-nitroso derivatives is not well understood. This studyexplores RDX transformation to its N-nitroso derivativesin mammalian GI tracts.

N-nitroso compounds represent a class of potent carcin-ogens. Some of them manifest carcinogenic effects at tracelevels (Gray et al., 1991). N-nitrosopiperidine (NPIP) anddinitrosopiperazine (DNPIP), for example, are establishedcarcinogens in mice, rats, and hamsters and are possiblehuman carcinogens (Preussmann and Stewart, 1984). Car-cinogenicity studies of NPIP and DNPIP in rats haveshown that both toxicants induce tumors in the esophagus,nasal cavity, liver, and stomach (Druckrey et al., 1967;Lijinsky and Reuber, 1981; Gray et al., 1991). MNX,DNX, and TNX are analogs of NPIP and DNPIP(Fig. 1). Toxicity studies of RDX metabolites are relativelyrare compared to that of the parent compound (Gonget al., 2001; Rosen and Lotufo, 2005; Mukhi et al., 2005;Best et al., 2006). Preliminary MNX and TNX toxicitystudies have shown reproductive effects in laboratory miceand soil invertebrates (Smith et al., 2006; Zhang et al.,2006a; Zhang et al., 2006b). George et al. (2001) have dem-onstrated mutagenicity of TNX using a modified salmo-nella bioassay (George et al., 2001). Thus, it is importantto understand whether RDX transforms to its N-nitrosoderivatives in mammals and the extent of such transforma-tion. Here, the GI tract is of interest because of its extensivebacterial activities. RDX transformations are expected tobe hastened under such conditions. The primary aim of thisstudy was to explore RDX transformation to its N-nitrosoderivatives in the GI tract of deer mice. Also, other toxicityendpoints such as food intake, body weight changes, organweights, and behavior abnormalities were also investigatedin deer mice, one of the most common woodland rodentspecies indigenous to North America (Turner et al., 2002;Presti et al., 2004).

2. Materials and methods

2.1. Chemicals

RDX used for dosing was obtained from Accurate Ener-getics (McEwen, TN, USA). The chemical was 99% pureand supplied in desensitized forms containing about15%–20% water by volume. For use in instrumental analy-ses, an RDX stock solution at a concentration of 1000 mg/lin acetonitrile (>99% pure) was purchased from Supelco(Bellefonte, PA, USA). Standards of MNX (>99% pure),DNX (59% pure), and TNX (>99% pure) were purchasedas solids from SRI International (Menlo Park, CA, USA).

2.2. Dose preparation

Deer mouse were exposed to RDX-contaminated food.Three concentrations (0, 10 mg/kg, and 100 mg/kg) wereprepared in mice chow. An estimated amount of RDXwas dissolved into acetone and sprayed onto Purina Certi-fied Rodent Chow� No. 5002 (Purina Mills, St. Louis,MO, USA). For control groups, an equal amount of ace-tone was sprayed onto chow as a vehicle control. Spikedchow was spread in a fume hood to allow acetone evapora-tion for 4 days before dosing started. RDX concentrationin chow was confirmed by gas chromatography with elec-tron-capture detection (GC–ECD) analysis after acetoneevaporation.

2.3. Animal treatments

Female virgin deer mice (Peromyscus maniculatus) (45days of age) from the Peromyscus Genetic Stock Center(University of South Carolina, Columbia, SC) were accli-mated for 7 days and then randomly assigned to control,low dose (10 mg/kg), and high dose (100 mg/kg) groups.They were housed three per cage with six replicate micefor each treatment group. Cages were located in an animalroom with temperature ranging from 18.3 to 25.6 �C, 25%–75% relative humidity, and 16:8-h light: dark cycle. Tap

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Fig. 2. Average body weight gains of deer mice. Error bars representstandard deviations of six replicates.

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water was used as drinking water. Mice, food and waterwere checked daily. The protocols for the use of animalsin this study were in compliance with Texas Tech Univer-sity Animal Use and Care Committee guidelines.

Deer mouse exposure to RDX-contaminated chowlasted nine days. Mice and chow were weighed before dos-ing and after dosing. Behaviors were also noted at 24 hintervals. At day 10, mice were euthanized by CO2 asphyx-iation and heart puncture. The intestinal tract, liver, kidney,and brain were removed, weighed, transported to our ana-lytical labs on dry ice, and stored at �20 �C pending analy-sis. The stomach and intestines were extracted separatelyfor RDX, MNX, DNX, and TNX determination.

2.4. Chemical analysis

The concentration of RDX and its N-nitroso deriva-tives, MNX, DNX, and TNX were determined in spikedchow, in the stomach, and in the intestine (including con-tent and tissue). The protocol for sample extraction,cleanup, and analysis using GC–ECD was according tothe procedure reported previously with necessary modifica-tions (Pan et al., 2005; Zhang et al., 2005). Briefly, eachsample was weighed and homogenized with dried Na2SO4

to dehydrate. The sample-Na2SO4 mixture was then loadedinto an extraction cell. All extractions were performedusing a Dionex ASE 200 extractor (Salt Lake City, UT).Each extraction cycle included a five min preheatingstep, followed by a five min static extraction with 100%acetonitrile at constant temperature (100 �C) and pressure(1500 psi). Extracts (15–20 ml/sample) were then purgedfrom cells into glass collection vials and reduced to 2–3 ml using rotary evaporation in preparation for cleanupusing a styrene-divinybenzene (SDB) cartridge. SDB car-tridges were conditioned with acetonitrile (2 · 3 ml). Sam-ples were then loaded, and eluates were collected. SDBcartridges were then rinsed with acetonitrile (3 · 2 ml),and eluates were collected. The extract volume (8–10 ml)was reduced to less than 2.0 ml using nitrogen evaporation.The final volume was adjusted to 2 ml with acetonitrile, fil-tered through a 0.45 lm membrane filter (Millipore, Bed-ford, MA, USA) into an autosampler vial, and stored(�20 �C) until GC–ECD analysis.

Standards and samples were analyzed using an HP 6890Series GC coupled with electron-capture detector (Agilent,Palo Alto, California, USA). Separation was performed ona 30-m · 0.25-mm-i.d · 0.25 lm film thickness HP-5 col-umn (Wilmington, DE, USA). Helium carrier gas wasmaintained at 100 cm/sec. The oven temperature programwas as follows: 90 �C hold for 2 min, ramp to 130 �C at25 �C/min, then ramp to 200 �C at 10 �C/min, then rampto 250 �C at rate of 25 �C/min. Injector temperature was170 �C and detector temperature was 270 �C. The carriergas flow rate and the injector temperature were the criticalparameters (Pan et al., 2005). The septum and inlet linerwere replaced after every 50 injections. The method detec-tion limits were around 10 lg/kg for each compound.

2.5. Data analysis

Data including chow weight, body weight, absoluteorgan weight, relative organ weight (organ weight/bodyweight), and toxicant concentrations in chow and GI tractwere processed using standard statistical software SPSS(SigmaPlot, Version 8.0, and SigmaStat, Version 2.03, SPSS,Chicago, Illinois, USA). Analysis of variance (ANOVA)was used for comparing the means of different treatmentgroups. If there was a significant difference among groups,least significant difference (LSD) multiple comparisonswere conducted to compare the mean of each group.

3. Results

3.1. Dosed food concentrations, food consumption and

body weights

Dosing chows were sampled, extracted and analyzedusing GC–ECD to verify the actual RDX concentrations.RDX concentrations in chow were 11.7 ± 0.9 mg/kg(n = 3) for the low dose group (nominal 10 mg/kg group)and 120.4 ± 4.3 mg/kg (n = 3) for the high dose group(nominal 100 mg/kg group). No detectable RDX wasfound in control chow.

Fig. 2 shows dose-dependent decreases in the averagebody weight gain after 9 days of exposure. Compared tothe control group, body weight gain in female deer micewas reduced by 35% and 53% for 10 mg/kg dose groupand 100 mg/kg dose group, respectively. However, suchdifference were not statistically significant (p = 0.13). Com-pared to the control group, food consumption was slightlydecreased with reductions of 3.5% and 8.6% for 10 mg/kgdose group and 100 mg/kg dose group, respectively. Thereis no significant difference in food consumption.

3.2. N-nitroso compounds in GI

The stomach and the intestine were extracted separatelyfor RDX, MNX, DNX, and TNX determination. Table 1shows that significant amounts of N-nitroso derivatives,MNX and DNX were found in the stomach and the intes-tine. In the stomach, average concentrations of MNX were

Table 1Detection of N-nitroso derivatives of RDX in deer mice GI tract (lg/kg) (n = 6 for each treatment group)*

Dose (mg/kg) Stomacha,b Intestinea,b

RDX MNX DNX TNX RDX MNX DNX TNX

0 ND** ND ND ND ND ND ND ND10 7324a (4824–11121) 85a (40–183) 217a (107–441) ND 18a (12–29) 14a (3–63) 40a (26–63) ND100 48622b (28804–82079) 1318b (963–1803) 498b (338–733) ND 22a (5–92) 43b (19–100) 53a (24–115) ND

a Geometric Mean (95% confidence intervals).b Parent and selected reductive transformation products were measured. Mass balances are not possible since oxidative and ring cleavage products were

not quantified.* Different letter in the same column represents a significant difference within the same compound at the same site.

** ND represents non-detected

X. Pan et al. / Chemosphere 67 (2007) 1164–1170 1167

85 lg/kg for the low dose group and 1318 lg/kg for thehigh dose group, while DNX concentrations were 217 lg/kg for the low dose group and 498 lg/kg for high dosegroup. In the intestine, average MNX concentrations were14 lg/kg for the low dose group and 43 lg/kg for the highdose group, while DNX concentrations were 40 lg/kg forthe low dose group and 53 lg/kg for the high dose group.No TNX was detected in the stomach or the intestine. Asmall amount of MNX was founded in the dosed food(�0.2% of the initial RDX concentration), probably dueto the impurities produced during manufacture. However,the ratio of N-nitroso derivatives to RDX in both stomachand intestine was significantly higher than that in the dosedfood (p < 0.01, Fig. 3). No detectable DNX and TNX werefounded in the dosed food.

3.3. Organ weights

Fig. 4 indicates general dose-dependent increases inorgan weights, especially in kidney and liver. Kidney man-ifested the most significant weight increase among the threeorgans investigated. There was a highly significant increase(p = 0.003) in absolute kidney weight between control andhigh dose group. Although absolute kidney weight of lowdose and high dose group were not distinctly different(p = 0.07), the p-value approached the critical value(p = 0.05). Absolute liver weight significantly increased

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Fig. 3. (A) Ratio of MNX concentration to RDX concentration in thedosed food, the stomach, and the intestine; and (B) Ratio of DNXconcentration to RDX concentration in the dosed food, the stomach, andthe intestine (note that DNX was not detected in food, bar on the graphrepresents one half the detection limit for that analyte).

(p = 0.04) between control and high dose group, but theincrease was not significant between low dose group andhigh dose group (p = 0.10). There was a general trend ofdose-dependent absolute brain weight gain; however, thegain was not statistically significant (p = 0.10 for controlversus high dose group and p = 0.12 for low versus highdose group). Relative organ weight gains were more evi-dent; significant increases in kidney and liver weight wereobserved between control and the high dose group as wellas between the low and high dose groups (p = 0.0008–0.04). Relative brain weight significantly increased betweencontrols and the high dose group (p = 0.046). There was nosignificant difference in organ weights (both absolute andrelative) between controls and low dose groups.

3.4. Behavioral observations

Deer mice behavior was observed daily. No grosslyabnormal behavior was observed within the first threedays. At day 4, stereotyped behaviors (jumping, backwardsomersaulting) were observed in the low dose group butnot in control and high dose group. Stereotyped behaviorsof low dose group mice persisted for the remainder of theexposure. Compared with control mice, reduced activitywas noted for the high dose group from day 5 on.

4. Discussion

Although environmental RDX biodegradation has beenwell studied for bioremediation purposes (McCormicket al., 1981; Kitts et al., 1994a; Young et al., 1997a; Hawariet al., 2000b; Adrian and Arnett, 2004), RDX transforma-tion in animals has been rarely studied. RDX biodegrada-tion pathway and products vary when using differentremediation strategies. Common transformation productsinclude N-nitroso derivatives (such as MNX, DNX, andTNX) hydroxylamino derivatives (such as hydroxy-lamino-dinitroso-RDX), and hydrolytic ring cleavageend-products such as formaldehyde, nitrous oxide, andhydrazines (McCormick et al., 1981; Kitts et al., 1994b;Young et al., 1997b; Hawari et al., 2000a; Adrian andArnett, 2004). Due to the potent carcinogenic characteris-tics of most N-nitroso compounds, concerns have arisenregarding the consequential RDX reduction to N-nitroso

Absolute kindey weight

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Fig. 4. Absolute and relative organ weights of deer mice exposed to RDX. Relative organ weights were calculated as: absolute organ weight/body weight.Error bars represent standard deviations of six replicates. Columns labeled with different letter represents significant difference.

1168 X. Pan et al. / Chemosphere 67 (2007) 1164–1170

derivatives in mammals. Although it is not the primaryRDX degradation pathway, our findings indicated that sig-nificant dose-dependent amounts of N-nitroso derivativesof RDX (MNX and DNX) are produced in the GI tractof deer mice. DNX, which was not found in the RDX-dosefood, was also detected in both stomach and intestine, withan average concentration as high as 498 lg/kg in stomach.The appearance of high concentrations of MNX and DNXin the stomach followed by concentration decreases inRDX, MNX, and DNX in the intestine also suggested thatthe reductive transformation of RDX to its N-nitrosoderivatives mainly occurs in the stomach. Bacteria in thestomach that can produce nitroreductase such as Esche-richia coli may play a role in this reaction. Peterson et al.reported that E. coli can reduce nitrofurazone via type Iand type II nitroreductases (Peterson et al., 1979). Mem-bers of the Enterobacteriaceae and Clostridium which havebeen proven to biotransform RDX to N-nitroso com-pounds in vitro are also predominant species in the largeintestine and colon of mice (Lee et al., 1968), so we initiallyexpected that bacteria in large intestine and colon may play

a major role in the reaction. However, due to the highRDX concentration in the stomach, the absolute mass ofN-nitroso products is larger in stomach. The averageRDX detected here in the intestine is in low ppb level(Table 1). However, the ratio of N-nitroso metabolites toRDX in the intestine is higher than that in the stomach(p < 0.01, Fig. 3), indicating that the reductive transforma-tion of RDX to its N-nitroso metabolites is progressivelymore efficient as materials move through the GI tract.

Reduction in body weight gain in dose female deer mice(35–53%) appears more pronounced than the 24% depres-sion in body weight gains observed for Fisher F344 ratsexposed to 125 mg/kg RDX (Levine et al., 1981). Food con-sumption was slightly decreased in the dose groups (3.5–8.6%). Similar effects were previously observed (Levineet al., 1981). Elevations in organ weight were observed indosed animals. For kidney and liver, there are significantdifferences in relative organ weight between low dose groupand high dose group, and highly significant differencebetween control group and high dose group. This indi-cated that renal and hepatic toxicity are likely to occur at

X. Pan et al. / Chemosphere 67 (2007) 1164–1170 1169

100 mg/kg RDX exposure for deer mice. The increase inrelative brain weight was not accompanied by change inabsolute brain weight. This suggested that decrease in thetotal body weight was responsible for the relative increasein brain weight (Smith et al., 2006).

Whether these toxic effects were caused by the parentcompound (RDX) or by the transformation products suchas MNX, DNX, and TNX was uncertain. Nevertheless,studies are emerging to assess the toxicity of these N-nitroso derivatives (Meyer et al., 2005; Smith et al.,2006). A comparative up- and -down oral toxicity studyrevealed the decreased lethality as RDX to its N-nitrosoderivatives (Meyer et al., 2005; Smith et al., in press).TNX was found mutagenic in Salmonella histidine rever-sion assay (George et al., 2001). Low dose aqueous TNXdosing (10 and 100 lg/l) in deer mice revealed toxiceffects including body weight reduction, relative brainweight elevation, and a dose-dependent offspring mortalityincrease (Smith et al., 2006). Current MNX and TNX tox-icity studies in soil invertebrate show a dose-dependentinhibition pattern in earthworm growth (Zhang et al.,2006b) and cricket egg hatching (Zhang et al., 2006a),and these N-nitroso derivatives of RDX appear more toxicthan their parent compound to earthworms (Zhang et al.,2006b).

Carcinogenic nitrosamines are involved in the etiologyof several types of cancer, including hepatic, gastric, esoph-ageal, nasopharyngeal, colon, and bladder cancer (Druck-rey et al., 1967; Lijinsky and Reuber, 1981; Gray et al.,1991; Mirvish, 1995). Endogenous formation appears tobe the most potent source of human exposure to nitrosa-mines following the reaction between nitrosating agentsand nitrogenous substrates (Ohshima and Bartsch, 1981).Actually, DOD suggests that metabolic activation ofRDX could hold the key to understanding the causes ofincreased instances of adenomas in 48 month chronic dos-ing studies (USDOD, 2001). However, details of metabolicinduction were not evaluated during their study. The pres-ent study provides basic data concerning RDX transforma-tion to its N-nitroso derivatives in vivo of mammals. Thisinformation may be useful in efforts to elucidate the poten-tial carcinogenic effects of RDX.

Acknowledgements

This work was funded by the Strategic EnvironmentalResearch and Development Program (SERDP), ProjectNo. CU1235. We would like to think Dr. M. Hooper forlogistical advice and T. McBride for assistance.

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