Oxazepam is mutagenic in vivo in Big Blue® transgenic mice

B.S. Shane, J.G. deBoer1, B.W. Glickman1 and M.L. Cunningham2,3

Institute for Environmental Studies, Louisiana State University, Baton Rouge, LA 70803, USA,
1 Centre for Environmental Health, University of Victoria, Victoria, BC V8W 3N5, Canada and
2 Environmental Toxicology Program, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although oxazepam (Serax®), a widely used benzodiazepine anxiolytic, does not induce gene mutations in vitro or chromosomal aberrations in vivo, it was found to be a hepatocarcinogen in a 2 year bioassay in B6C3F1 mice. Thus, it was of interest to determine whether this carcinogen is mutagenic in vivo. Male B6C3F1 Big Blue® transgenic mice were fed 2500 p.p.m. oxazepam or control diet alone for 180 days and killed on the next day. The mutant frequency (MF) of lacI in control mice was 5.02 ± 2.4x105, whereas the MF in the oxazepam-treated mice was 9.17 ± 4.82x10–5, a significant increase (P < 0.05). Correction of the mutant frequency of lacI from the oxazepam-treated mice for clonality resulted in a decrease in the mean mutant frequency to 8.15 ± 2.54x10–5. Although the mutant frequency difference was small, sequencing of a random collection of the mutants from each oxazepam-exposed mouse showed a significant difference (P < 0.015) in the mutation spectrum compared with that from control mice. In the oxazepam-exposed mice, an increase in G:C->T:A and G:C->C:G transversions and a concomitant decrease in G:C->A:T transitions were observed. Clonal expansion of mutations at guanines in 5'-CpG-3' sequencing contexts at three sites was noted. It is postulated that some of the mutations found in the oxazepam-derived spectrum were due to oxidative damage elicited by induction of CYP2B isozymes as the result of chronic oxazepam administration. This study demonstrates that the in vivo Big Blue® transgenic rodent mutation assay can detect mutations derived from a carcinogen that did not induce gene mutations in vitro or micronuclei in mouse bone marrow. Moreover, the sequencing of the recovered mutants can distinguish between the mutation spectrum from treated mice compared with that from control mice, thereby confirming the genotoxic consequences.

Abbreviations: DMN, dimethylnitrosourea; ENU, ethylnitrosourea; MF, mutant frequency; NTP, National Toxicology Program.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oxazepam (Serax®), a benzodiazepine compound, is a central nervous system depressant which is widely prescribed for the treatment of anxiety. Most clinically useful drugs for this purpose consist of the 1,4-benzodiazepine moiety, comprised of two aromatic rings and a seven-membered heterocycle. One of the aromatic rings is fused to the seven-membered ring, and contains a chloro-substituent or some other electronegative group. All clinically important derivatives contain a 5-aryl, or a 5-cyclohexenyl group. Oxazepam is the active metabolite of a number of related benzodiazepines including diazepam (Valium), and chlordiazepoxide (Librium) which have been in use for >=40 years. The use of benzodiazepines in the general population has been reported to be as high as 7% in the US.

Although it is weakly clastogenic in vitro in Syrian hamster embryo cells and L5178Y mouse cells, oxazepam does not induce chromosomal aberrations in mouse peripheral blood erythrocytes, suggesting that it is not clastogenic in vivo (1,2). Furthermore, although oxazepam does not induce gene mutations in vitro in the Ames test or sister chromatid exchanges in Chinese hamster ovary cells, its ability to induce gene mutations in vivo has remained undetermined. Despite the lack of in vivo mutagenicity observed to date, two studies have shown oxazepam to be carcinogenic in mice. Fox and Lahcen (3) observed liver tumors in oxazepam-treated Swiss–Webster mice during the course of reproductive toxicity studies (3). More recently, liver neoplasia was documented in both sexes of Swiss–Webster and B6C3F1 mice in a 2 year feeding study undertaken by the National Toxicology Program (NTP) (1). In this study, exposure of male B6C3F1 mice to 0, 125, 2500 and 5000 p.p.m. of oxazepam resulted in hepatocellular carcinoma in 47, 38, 100 and 100%, respectively, of the mice (1,4). The relevance of the tumorigenicity of oxazepam in rodents to human health is unknown. Several mechanisms have been proposed to explain the tumorigenicity of carcinogens with properties similar to oxazepam. These include stimulation of cell division by mitogenesis (5), compensatory cell division as a result of cytotoxicity (6) and the interruption of cell–cell communication (7). Rapid cell division could shorten the length of the cell cycle and accelerate the G1, G2 or S phases, thus reducing the time for repair of DNA lesions and decreasing the fidelity of DNA replication (8,9). Cohen and Ellwein (5,10) have proposed that rapid cell division could increase the amount of unprotected DNA exposed to endogenous reactive oxygen species, which are known to be mutagenic. Evidence for any of these hypotheses is lacking. Oxazepam is mitogenic but not cytotoxic at the doses used in the NTP bioassay and stimulates cell proliferation only during the first 3 weeks of feeding (11). Liver:body weight ratios remained elevated throughout the 2 year NTP study due to hypertrophy and hyperplasia (11).

Although oxazepam is tumorigenic in mice, there is no evidence that it causes point mutations in growth regulating genes in rapidly dividing cells. In fact, it was shown that the mutation specificity in the H-ras gene at codon 61 and other frequently mutated sites in hepatic tumors does not differ in oxazepam-treated mice from those in control mice (12). However, the highest percentage of tumors (35%) with H-ras mutations in the oxazepam study was found in the group receiving the lowest dose of chemical (125 p.p.m.). Rather than being an initiating agent, oxazepam could promote the proliferation of cells harboring a mutation in other important genes besides H-ras.

The Big Blue® transgenic rodent mutagenesis model has mainly been used to measure mutations following short-term exposure of rodents to high doses of mutagens by gavage or i.p. injections (13,14). Only a few studies have used longer treatment regimens of less potent mutagens. For example, Big Blue® mice have been shown to be sensitive to the administration of butadiene by inhalation for 4 weeks (15), and to 2,4-diaminotoluene included in the diet for 3 months (16). In the latter study, we demonstrated that this Big Blue® in vivo mutagenesis model could distinguish between a mutagenic carcinogen and a mutagenic non-carcinogen (16). Although there is some evidence to suggest that oxazepam may not be mutagenic in vivo, the evidence is based on only one test, which was not in the target tissue for carcinogenicity. Accordingly, it was of interest to determine if oxazepam is mutagenic in mouse liver. For this study, a longer term continuous feeding study was chosen in order to increase the sensitivity of the assay and to more closely mimic the dosing regimen used in the standard NTP bioassay. Big Blue® mice were fed a diet containing 2500 p.p.m. oxazepam for 180 days. The mutant frequency (MF) at lacI was determined by recovering the genomically integrated {lambda}LIZ shuttle vector using an in vitro packaging reaction followed by infection of an appropriate Escherichia coli host. Blue mutant plaques were scored against a background of clear non-mutant plaques. An unbiased selection of mutants from each of the mice was sequenced and the mutation spectrum compared with that generated from untreated control mice.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and treatment
Age-matched 6-week-old male B6C3F1 transgenic mice bearing multiple copies of the {lambda}LIZ shuttle vector harboring the lacI gene stably integrated into chromosome 4 of their genome (Big Blue® mice), were obtained from Taconic Farms (Germantown, NY). The mice were randomly housed, five per cage, and were quarantined for 7 days before treatment was initiated. All animals were housed in rooms maintained on a 12 h on–off light cycle. For untreated control and oxazepam-exposed animals, powdered NIH-07 feed containing either 0 or 2500 p.p.m. oxazepam, respectively, was provided ad libitum for the length of the study. Eight animals were assigned to each treatment group. The day after feeding of the drug was halted the mice were weighed and killed by CO2 asphyxiation. The liver and kidney were harvested; the liver was weighed and both tissues flash frozen in liquid nitrogen and stored at –80°C until analysis. Positive control animals received five consecutive daily i.p. injections of 6 mg/kg of dimethylnitrosamine (DMN) in saline and were killed by CO2 asphyxiation 15 days following the last injection.

Isolation of DNA
DNA was isolated using the RecoverEase protocol (Stratagene Cloning Systems, La Jolla, CA). Briefly, 50 mg of liver was homogenized in 8 ml of ice-cold buffer (1.75 g Na2HPO4, 8.0 g NaCl, 0.2 g KH2PO4, 20 ml of 0.5 M EDTA, pH 8.0) with a `B' pestle two to three times until disaggregation and then with an `A' pestle for eight strokes to rupture the cells and release the nuclei. The homogenate was filtered though a nylon mesh filter and centrifuged at 1000 g for 10 min at 4°C. The supernatant was decanted and the tube dried to remove the disaggregation buffer. The pellet was incubated at 50°C for 45 min in the presence of digestion buffer containing RNAceIt (20 µl/ml buffer) (Stratagene Cloning Systems) and proteinase K. The contents of the tube were dialyzed for 48 h by placing the DNA extract on the wetted surface of a membrane floating on 10 mM Tris/1 mM EDTA buffer pH 7.4. The viscous DNA was harvested and stored at 4°C until packaging.

Packaging and plating of DNA
Excision and packaging of the shuttle vector from genomic DNA was performed using {lambda} phage packaging extract (Transpack; Stratagene Cloning Systems) according to the Stratagene Big Blue® Instruction Manual with modifications (17). The titer of the rescued phage was estimated by plating serial dilutions of the packaged phage. For plating, a volume of phage equivalent to 10 000 plaque forming units (p.f.u.) was added to 2 ml of E.coli SCS-8 cells (Stratagene Cloning Systems) in 10 mM MgSO4. Top agarose containing 1.5 mg/ml of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) was added, and the contents poured onto 25x25 cm assay trays containing 250 ml NZY amine agar. The phage was re-titered on the day of plating. Trays were incubated for 18 h at 37°C and examined for blue mutant plaques. Plaques that were entirely blue in appearance were assumed to arise from mutational events occurring in vivo. Sectored plaques, which were identified in the initial plating as plaques containing both blue and clear areas (with <50% to a pinpoint of blue), were omitted from the calculation of the MF. The number of p.f.u. plated on the trays was calculated based on the titer obtained from triplicate plates prepared at the time of plating for mutants. Each mutant plaque was cored from the plate, placed in 0.5 ml SM buffer (100 mM NaCl, 8 mM MgSO4·7H2O, 50 mM Tris buffer pH 7.5, 0.01% gelatin) and the phenotype confirmed by replating. The MF was determined by dividing the number of mutant plaques by the total number of p.f.u. evaluated from each animal. A Mann–Whitney test was performed to assess significant differences in the MF between the treatment and control groups. Based on the sequencing data, the MF of the oxazepam-treated mice was corrected for clonal expansion and the data re-evaluated statistically for differences from the MF in the control mice.

Sequencing of the mutants
DNA sequencing of the confirmed oxazepam mutants was performed using a DNA Long Read IR 4200 automated sequencer (Licor Biotech, Omaha, NE). The lacI gene was amplified from the bacteriophage by PCR in a 100 µl PCR reaction mixture containing 5 µl of phage, 10 pmol of each of two primers located at –215 to –235 (5'-AGCGTCGATTTTGTGATGCT-3') and 1137–1354 (5'-CGCTATTACGCCAGCTGG-3'), 200 mM dNTPs and 5 U Taq DNA polymerase. The resulting 1400 bp fragment, which included the lacI promoter and the lacZ operator region, was purified on a Wizard PCR Prep DNA purification column (Promega Corporation, Wisconsin, MI) (18). Cycle sequencing of the DNA was performed on 100 ng of template with 2.5 pmol of forward sequencing primer, located at positions –103 to –82 (5'-CTGTGGATAACCGTATTACCGC-3') and reverse sequencing primer, located at positions 1187 to 1208 (5'-TCCGCTCACAATTCCACACAAC-3'). Sequencing was done in a single reaction with the two primers each labeled with a different fluorescent dye, allowing for the reading of the sequence from two directions at once. Sequence data were analyzed statistically to determine differences in the classes of mutations in the oxazepam versus control mutants using hypergeometric methods (19).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since oxazepam is known to be a mitogen, liver:body weight ratios were measured in all the mice at time of killing, 180 days after feeding began. This ratio, ~190% compared with controls, was significantly higher (P < 0.01) in the livers of treated mice compared with control animals (data not shown), indicating that the liver underwent mitogenesis and remained enlarged throughout the remaining 5 months of feeding. This increase in liver weight is consistent with a previous study which found an increase in liver weight during the first 3 months of exposure to oxazepam (11).

To assess whether oxazepam is a mutagen in vivo, the frequency of the lacI mutants was determined in the livers of nine male B6C3F1 control mice and eight mice treated for 180 days with 2500 p.p.m. of oxazepam in the diet. In the oxazepam-exposed mice, the MF of lacI was 9.17 ± 4.82x10–5 compared with 5.02 ± 2.4x10–5 in the control mice (Table IGo), a statistically significant increase (P < 0.05). It is noted that the MF of lacI from mouse no. 2 from the oxazepam-treated group was significantly higher than the MF from the other mice. As shown below, this higher MF in mouse no. 2 can be explained by the finding that 46% (13/28) of the mutants from this mouse had a G->A mutation at base pair 56, and 14% (4/28) of the mutants had a G->A transition at base pair 180. We are currently comparing these individual results in another target gene in this system, cII.


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Table I. Mutant frequency of lacI in the livers of control, oxazepam and DMN-treated Big Blue® mice
 
Since clonal expansion (see below) was potentially a major contributor to the MF in this mouse and also a contributor in mice nos 4, 5 and 8 of the oxazepam-treated group, the MF was corrected for clonal expansion (Table IGo). This MF correction reduced the MF in mouse no. 2 from 20.80x10–5 to 13.2x 10–5 and, with clonal correction in the other three mice (Table IGo), resulted in a corrected MF for the oxazepam-treated group of 8.15 ± 2.54x10–5. This decrease in variance resulted in an increase in the statistical significance (P < 0.04) between the treated and control groups. The MF of the DMN-treated mice, which were included as a positive control group, was 28.6 ± 6.3x10–5.

Classes of mutations from the oxazepam-treated mice
Of the 184 mutants collected from the eight oxazepam-treated mice, 132 were randomly chosen and sequenced. A minimum of 12 mutants from each mouse was analyzed. Mutations were identified in 130 of the mutants. Of these, 109 mutants (83.8%) were base pair substitutions, with 101 of them (92.7%) at G:C sites and 28 generated stop codons. There were 10 frameshift mutations, eight deletions and three complex mutations. In contrast to findings in the historical control mice, where ~60% of the mutations occurred in the first 177 bp of the gene (DNA-binding domain), only 43% of the mutations in the oxazepam-treated mice occurred in this domain.

Using the method of Adams and Skopek (19), a significant difference (P < 0.015) in the corrected oxazepam-spectrum compared with the historic control spectrum (20) was obtained (Table IIGo), i.e. the resulting spectra were distinct. Specifically, an increase in the frequency of transversions was noted in the oxazepam-treated mice compared with the control mice. G:C->T:A transversions increased from 18.4 to 28.5% and G:C->C:G transversions increased from 3.9 to 12.4% which translated to 3.3- and 7.7-fold increases in the frequency of these mutations, respectively. A corresponding reduction in G:C->A:T transitions from 48.9 to 34.3%, was observed. Interestingly, the percentage of G:C->T:A transversions at 5'-CpG-3' sites increased from 38.5% in the control mice to 66.7% in the treated mice. Amongst the non-base pair substitutions, only the frequency of deletions was altered by oxazepam-treatment, resulting in a 5.6-fold increase (Table IIGo).


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Table II. Classification of lacI mutations recovered from oxazepam-fed Big Blue® mice
 
The 109 base pair substitutions recovered after oxazepam treatment involved 63 sites. Forty-six of these mutations were at unique sites and the remainder mapped to 17 sites. Of these sites, more than one type of base substitution was recovered at three sites while multiple occurrences of the same substitution were identified at 14 additional sites (Table IIIGo). The majority of sites at which multiple mutations were recovered likely represent true hotspots as the same mutations were recovered at these sites in different mice.


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Table III. Multiplicity of lacI mutations recovered at the same site from oxazepam-fed Big Blue® mice
 
A closer examination of the 14 sites at which more than one mutation was observed, revealed that, with the exception of positions 134, 260 and 953, all were in 5'-CpG-3' contexts. In the cases of positions 56, 86, 129, 134, 150, 198, 260, 329, 528, 606, 882 and 953, the same mutations were recorded in two or three mice. In two mice, nos 2 and 8, clonal expansion appeared to be the cause of 13 G->A mutations at position 56, whereas four G->A mutations at position 180 and four C->G mutations at position 260 were found in mice nos 2 and 4, respectively.

All of the frameshift mutations recovered following oxazepam treatment involved single base pairs (Table IVGo), with six of them in homopolymeric runs. The occurrence of these six frameshift mutations can be explained by slippage. The single base deletions at base pairs 371 and 784, and the single base insertions at base pairs 518 and 695 cannot be explained by the conventional slippage model. Deletions of four base pairs at the bacterial hotspot 620–632 was noted in two mutants (Table VGo), while a different four base pair deletion at base pair 738 was obtained in one mutant. Larger deletions, from 9 to 122 bp, occurred in five mutants (Table VGo). Four of these mutations had repeated sequences ranging in length from 2 (mutant 371) to 7 bases (mutant 735) at their endpoints. In these cases, the deletions included one of the repeat sequences; thus, they could be explained by the slippage model. Complex changes were recorded in three mutants: in two cases, a different deletion of 24 bp occurred with a concomitant base pair change at base pair 41 in mutant 775, and the insertion of a triplet TCT at the deletion point in mutant 726. This latter insertion may have been due to the fact that a TCT triplet is present three bases downstream of the junction point. In the third complex mutant (865), an adenine was deleted between bases 135–139 and a transition (A->G) was found at base 139. No double substitutions were recorded.


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Table IV. Frameshift mutations recovered from oxazepam-fed Big Blue® mice
 

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Table V. Deletion mutations recovered from the liver of oxazepam-treated Big Blue® transgenic mice
 
Clonal expansion
Clonal expansion is usually defined as the recovery of multiple mutations at the same site in the same tissue of the same animal. When identical mutations are noted under these conditions, conservative correction counts these events as a single event. Clonal expansion is of particular concern in rapidly dividing tissues, a situation which occurs following mitogen stimulation. Thus, the 13 and five G->A transitions which were recovered at position 56 from mice nos 2 and 8, respectively, constitute a possible clonal event. It is also possible that clonal expansion occurred in mice nos 2 and 4 resulting in four G->A transitions and four G->C transversions at base pairs 180 and 260, respectively (Table IIIGo). Not all the identical mutations recovered from the same tissue from the same animal need represent clonal events but the conservative explanation for these mutations is their formation through clonal expansion. In the case of chronic exposure to oxazepam, however, fixation of many mutations throughout the exposure period of 6 months could certainly have occurred at base pairs 56 and 180, which are frequently mutated. It is likely that the four G->C transversions at base 260 were the result of a clonal expansion as this particular mutation is rare in Big Blue® mice (20).

Transcribed versus non-transcribed strands
Analysis of the base pair substitutions on the transcribed versus non-transcribed strands showed that 64 (58.7%) of the base pair substitutions from the oxazepam-treated mice were on the transcribed strand and 44 (41.3%) were on the non-transcribed strand. When the total number of unique base pair substitutions were examined, 52.2% (35/67) and 47.8% (32/67) were found on the transcribed and non-transcribed strands, respectively. Thus, no obvious indication for strand bias was observed.

Sequence context
Of the 36 independent individual G:C->T:A transversions recovered from lacI in the treated mice, 30 (83%) were at 5'-PyGN-3' sequences, while six (17%) were at 5'-PuGN-3' sequences where `Py' is a pyrimidine, `Pu' is a purine and `N' is any of the four bases (analyses not shown). In contrast, in control mice, only 50% of the G:C->T:A transversions were in 5'-PyGN-3' sequences. More than 69% (9/13) of the G:C->C:G transversions were at 5'-PyGN-3' sequences while 31% (4/13) were at 5'-PuGN-3' sequences. Of the 27 unique G:C->A:T transitions, 19 (70%) were at 5'-PyGN-3' sequences with 18 (95%) of these transitions in 5'-CGPy-3' contexts, and eight (30%) were at 5'-PuGN-3' sequences. The sequence context of G:C->A:T transitions differed in the control spectrum; specifically, 90% of the GC->AT substitutions were in 5'-PyGN-3' sequence contexts. Thus, irrespective of the type of base pair substitution at guanine, the sequence context of 5'-PyGN-3' was favored over the 5'-GGN-3' sequence context in the oxazepam-treated mice, with >55% of these mutations occurring in a 5'-CGPy-3' sequence context.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The major objective of this study was to assess whether oxazepam, which does not induce gene mutations in Salmonella (1) and causes liver tumors in rodents after 2 years of feeding, was mutagenic in vivo in Big Blue® transgenic mice. The results show that oxazepam caused an almost 2-fold increase in MF (P < 0.05) in lacI in transgenic mouse liver. Sequencing of a representative sample of the mutants from all of the treated mice supports the hypothesis that oxazepam is mutagenic in vivo. The clonally corrected mutation spectrum from the oxazepam-treated mice was significantly different (P < 0.015) compared with the historic control spectrum (20) in B6C3F1 transgenic mice. The types and location of the mutations in the lacI gene suggested that some of the mutations arose via oxidative damage.

Oxazepam was administered at 2500 p.p.m. in the feed for 180 days. The choice of a subchronic regimen was based on earlier findings in which we had shown that administration of the genotoxin 2,4-diaminotoluene at 1000 p.p.m., a relatively high dose, resulted in an increase in MF of lacI in the liver of treated mice only after dosing for 90 days (16). With a shorter feeding period of 30 days, no significant increase in the MF was obtained in the 2,4-diaminotoluene-treated mice. The dose of oxazepam used (2500 p.p.m.) corresponds to the lowest dose used in the 2 year study that resulted in a highly significant increased liver tumor incidence (1). The results with oxazepam suggest that, indeed, carcinogens that do not form DNA adducts but are tumorigenic via a different mechanism may need to be administered chronically to elicit a mutagenic response in vivo.

The finding that oxazepam increased (P < 0.05) the lacI MF in liver by 2-fold is interesting in light of the fact that it is considered to be a weak clastogen. Oxazepam does not form DNA adducts nor is it mutagenic in vitro; it is only clastogenic in cell cultures in vitro but not in vivo (21). Previously, it had been shown that oxazepam is a potent mitogen inducing hepatocellular proliferation, albeit for >1 month in a long term feeding study of 90 days (11). Cellular proliferation in target organs could contribute to an elevated MF in Big Blue® mice as a result of clonal expansion of either spontaneously or chemically induced mutated cells. This hypothesis is supported by the sequencing of the mutants in which it was shown that clonal expansion of mutations occurred at three different sites in each of four treated mice. It is unknown whether clonal expansion occurred in the control mice in this experiment as mutants from these mice were not sequenced; however, in a recent study (G.R.Stuart, Y.Oda, J.G.deBoer and B.W. Glickman, personal communication) it was found that clonal expansion did not contribute to the mutation spectrum of mutants collected from control mice that were 2 years old at time of killing.

The significance of a 2-fold increase in the MF in the oxazepam-treated mice compared with the control mice might be questioned as to its relevance to hepatocarcinogenicity in the mouse. However, sequencing of the mutants showed that the corrected oxazepam spectrum (Table IIGo) was significantly different (P < 0.015) compared with the historic control spectrum (20), thus giving credence to the relevance of the increase in MF found with oxazepam. The necessity for sequencing mutants when the increase in MF is marginal was demonstrated by deBoer et al. (22) who found that tris(2,3-dibromopropyl)phosphate increased the MF of lacI by only 50% in the kidney, the target tissue for carcinogenicity, and not significantly in the liver or stomach. Sequencing of the mutants from these tissues revealed a significant difference in the mutation spectrum only in the kidney of the tris(2,3-dibromopropyl)phosphate-treated mice compared with the controls. Thus, a doubling of the MF by oxazepam, a non-DNA reactive chemical in vivo (23), and the significant difference in the mutation spectrum in lacI can be considered as being biologically significant.

The significantly increased percentage of G:C->T:A and G:C->C:G mutations in the oxazepam-derived spectrum is consistent with the theory that oxygen radicals contributed to their formation as these lesions can arise from oxidative damage. Both transversions can occur as the result of oxidative damage to guanine residues resulting in the production of 8-oxoguanine (24,25). In the case of G->T mutations, the altered guanine residue may either mispair with adenine during replication or be excised leaving an abasic site as a premutagenic lesion. In either case, i.e. mispairing with adenine or the preferential insertion of an adenine opposite a non-instructional abasic site (the `A' rule) (26), a G:C->T:A transversion will result. G->C transversions arise from the mispairing of 8-oxoguanosine, with dGTP instead of dCTP (27). It has been pointed out that it is especially difficult to relate a particular reactive oxygen species with a particular mutation because more than 30 DNA adducts have been identified with oxygen radical damage (28). The type of radical that produces G->C transversions is presently debatable, although singlet oxygen has been implicated. More than one-third of the point mutations recovered from an SV40-based shuttle vector containing the supF gene as a mutational target following exposure to singlet oxygen were G:C->C:G transversions (29). Another major factor in mutation fixation is the role played by DNA polymerases. Loeb and co-workers have shown that in an in vitro system different lesions can result from superoxide radical damage depending on which mammalian polymerase is used to replicate the DNA (30).

It is difficult to determine whether the G:C->A:T transitions recovered at 5'-CpG-3' sites from the oxazepam-treated mice arose as the result of oxidative damage to guanine or from the spontaneous hydrolytic deamination of 5-methylcytosine which leads to the production of thymine, which in turn pairs with adenine resulting in a G:C->A:T transition (31,32). The proportion of G:C->A:T transitions in the oxazepam-treated mice at 5'-CpG-3' sites (64%) was less than the proportion of spontaneously derived mutations at these sites in untreated mice (75%). In non-CpG contexts, it is likely that the G:C->A:T transitions resulted from oxidative damage in the oxazepam-treated mice, but it is possible that a proportion of the G:C->A:T transitions at 5'-CpG-3' sites also result from oxidative damage. It is unknown whether the G->A mutations recorded at base 56 and base 180 in mouse no. 2 and at base 56 in mice nos 6 and 8 resulted from oxidative damage or from spontaneous events as both sites are in 5'-CpG-3' sequence context sites and are commonly mutated in untreated mice (20). It is possible, therefore, that neither of these mutations occurred as the result of oxazepam treatment. It should be pointed out, however, that base pairs 56 and 180 were mutated at high frequency in Big Blue® transgenic mice treated with the genotoxic agents, butadiene (15) and benzo[a]pyrene (33). In contrast to the mechanism of formation of the G:C->A:T transitions, the G:C->C:G transversion mutation at base pair 260, a rarely mutated site, likely arose from oxidative damage to guanine. This base is not in a 5'-CpG-3' sequence, and G->C mutations are usually reflective of oxygen radical related damage.

It is debatable whether the MF should be corrected for clonal expansion in studies in which the chemical is administered over extended periods of time. In chronic exposure regimens, identical mutations at the same site in one animal could result from clonal expansion of one mutation during the course of the study, or each mutation could be an unique event. In the present study, it cannot be determined whether the G->A mutations at base pairs 56 and 180 resulted from clonal expansion or from independent lesions. Oxazepam is mitogenic during the first 4 weeks of exposure (11); thus, mutations occurring during this time period could be clonally expanded. However, since the Big Blue® system captures accumulated mutations over time, in this study of 6 months, it would seem possible that identical mutations could arise in different cells, particularly at sites which are frequently mutated over the course of chemical exposure. In an attempt to assess the effect of cell division on clonal expansion, we have performed a partial hepatectomy on mice that were treated with ethylnitrosourea (ENU) before surgery. A 4-fold increase in MF in the ENU-treated partially hepatectomized mice was obtained compared with mice receiving only ENU. Even though division of the hepatocytes was stimulated after DNA damage had occurred, each randomly selected sequenced mutant had a unique mutation, and clonal expansion was not observed (34). In another study, no significant increase in clonal expansion of mutations was observed after 2 years, in the liver of untreated Big Blue® mice (Stuart et al., unpublished data). It would seem, therefore, that the practice of correcting MF data based on clonal expansion may be too conservative if the animal is exposed for extended periods to a chemical, since mutations could occur at any time. It is more likely that clonal expansion is involved when more than one mutant with the same mutation is recovered from the same animal if an acute exposure regimen was used and a large percentage of the cells in the tissue of interest replicated more than once before the tissue was harvested.

The mechanism by which oxazepam causes oxidative damage is indirect and likely involves the induction and increased activity of hepatic drug metabolizing enzymes. Data from our laboratory suggest that oxazepam is a potent inducer of CYP2B during the first 10 days of feeding (35). Whether the activity of this enzyme remained elevated throughout the 6 months of feeding oxazepam, is unknown. As superoxide anion is produced during cytochrome P450 metabolism, elevated levels and activity of CYP2B could potentially result in increased concentrations of superoxide anion radical. The spectrum obtained with oxazepam is consistent with DNA damage by oxygen radicals. Sustained induction of cytochrome P450 isozymes, especially the CYP1A, CYP2B, and CYP2E isozymes resulting in the production of reactive oxygen species have been implicated as mediators of hepatocarcinogenicity (36). Chronic induction of these cytochromes could also increase the bioactivation of exogenous (foodborne) compounds resulting in the generation of reactive oxygen species or electrophilic moieties, which in turn could increase the rate of mutagenesis and thus hepatocarcinogenesis (37,38).

In conclusion, this study demonstrates that the Big Blue® assay is able to identify in vivo mutations elicited by a chemical carcinogen that fails to induce consistent results in other mutagenicity assays. It appears as if oxidative damage was the primary cause of mutations at certain sites but clonal expansion of these lesions as well as those derived spontaneously may have contributed, in some cases, to the increase in MF observed in the oxazepam-treated mice. This study also illustrates that dietary exposure of chemicals is an appropriate route of administration for in vivo mutagenesis studies, and that longer exposure regimens should be considered in the design of studies assessing the in vivo mutagenicity of chemicals that are weak or negative in vitro. In addition, chemicals that are administered acutely and are not mutagenic in the Big Blue® assay should be retested under chronic conditions. We believe that the data obtained with oxazepam continues to validate this transgenic mouse model as a potential indicator of carcinogenic response, and suggest that the Big Blue® assay is also useful for mechanistic studies of the carcinogenicity of compounds that are not DNA reactive.


    Acknowledgments
 
The authors wish to acknowledge Daisy Smith for her technical contribution to this work and David Walsh, Ken Sojonky and Jana Kangas for their efforts in sequencing the mutants. Critical review of the manuscript by Drs Errol Zeiger (NIEHS) and David DeMarini (USEPA) is gratefully acknowledged. This study was partially supported by NIH CA72534-01 awarded to B.S.S.


    Notes
 
3 To whom correspondence should be addressed Email: cunning1{at}niehs.nih.gov Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 3, 1998; revised February 18, 1999; accepted March 17, 1999.