Mutagenesis

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Mutagenesis, Vol. 16, No. 5, 439-442, September 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press

Phenobarbital, oxazepam and Wyeth 14,643 cause DNA damage as measured by the Comet assay

Walter A. Deutsch^1,, Anu Kukreja, Barbara Shane^2, and Vijay Hegde

Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA 70808 and ² Integrated Laboratory Systems, Research Triangle Park, NC, 27709, USA

	Abstract

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Although phenobarbital, oxazepam and Wyeth 14,643 are carcinogens that do not form DNA adducts, they induce mutations in the Big Blue^® transgenic mouse model. The mutations produced by these compounds were predominantly GT and GC transversions that we suspect arose from oxidative damage to DNA. To test this, we employed the single cell electrophoresis (Comet) assay that detects alkali-labile lesions in cells sustaining DNA damage. Human myeloid leukemia K562 cells were treated with non-cytotoxic doses of the above compounds for 3 h, then placed on slides containing low melting point agarose. Cells were lysed, exposed to alkaline buffer, electrophoresed and analyzed by microscopy for the existence of DNA damage. Extensive DNA damage, most likely due to the existence of single- and double-strand breaks and apurinic/apyrimidinic (AP) sites, was observed in cells exposed to oxazepam (1 mM) and Wyeth 14,643 (0.5 mM). On the other hand, damage of this sort was not observed in cells exposed to phenobarbital (1 mM). However, the addition of S9 liver extracts to cells exposed in the presence of phenobarbital resulted in significant amounts of DNA damage. We conclude from these studies that two of the three compounds evaluated in this study mediate their mutagenic effects through oxidative stress, but that the mechanism of DNA damage caused by phenobarbital differs from that elicited by oxazepam and Wyeth 14,643.

	Introduction

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The mechanism by which non-DNA-reactive carcinogens cause mutations is for the most part unknown. Such compounds that we have studied in the past 2 years are oxazepam, phenobarbital and Wyeth 14,643. All of these compounds are extremely weak mutagens or clastogens; only phenobarbital is weakly positive in the Ames mutagenicity assay in strain TA1535 in the absence of S9 (Zeiger and Harworth, 1985; Albertini and Godke, 1992) and in the mouse lymphoma assay (Amacher and Turner, 1985; Myhr et al., 1985). None of the compounds are known to form DNA adducts as measured by the sensitive ³²P-post-labeling assay (Goel et al., 1985; Randerath et al., 1992; Whysner et al., 1998). Oxazepam and phenobarbital are weak clastogens; the former inducing chromosomal aberrations in Syrian hamster embryo cells and the latter in CHO cells (Gulati et al., 1985; National Toxicology Program, 1993). Wyeth 14,643 is classified as a peroxisome proliferator, compounds which are negative in the Ames assay (Glauert et al., 1984) and do not induce single-strand breaks in vivo in rat hepatocytes (Elliott and Elcombe, 1987). However, Wyeth 14,643 has been shown to be a clastogen at near toxic doses (Lefevre et al., 1994)

Despite their extremely weak mutagenicity in vitro, the three compounds are carcinogenic in 2 year bioassays in mice (Fox and Lahen, 1974; International Agency for Research of Cancer, 1987; National Toxicology Program, 1993). The mechanism of tumorigenesis is unknown, but recently we showed that the three compounds are mutagenic in vivo, producing either an increase in the mutant frequency or a significant change in mutation spectrum at two transgenic loci in Big Blue^® mice. Our findings at the lacI locus suggested that these compounds were mutagenic as a result of oxidative stress elicited by chronic feeding. In the case of phenobarbital and oxazepam we postulated that the up-regulation of specific cytochrome P-450s, of the 2B family (Waxman and Azaroff, 1992; Griffin et al., 1996), were producing oxygen stress through generation of superoxide radicals. These in turn led to the production of hydroxyl radicals, resulting in the formation of 8-oxodeoxyguanine that results in GCTA transversions. Chronic feeding of oxazepam and phenobarbital up-regulates CYP2B (Griffin et al., 1996). Wyeth 14,643, on the other hand, induces CYP4A as well as enzymes involved in lipid oxidation. Oxygen stress could be generated by either one or both of these pathways. Although both oxazepam and Wyeth 14,643 increased the mutation frequency at both the lacI (Shane et al., 1999a, 2000) and cII transgenes (Singh et al., 2001), this was not the case with phenobarbital. Nevertheless, phenobarbital significantly altered the mutation spectra at both loci, whereas oxazepam and Wyeth 14,643 only altered the mutation spectrum at lacI. These findings suggest that the three compounds might be causing mutations via oxidative stress but that the mechanisms were different. To investigate whether this was the case, we exposed human myeloid leukemia cells to the three compounds and measured DNA damage using the Comet assay, which reveals single- and double-strand breaks and baseless sites in DNA. All three compounds are shown to cause DNA damage, although phenobarbital only does so in the presence of S9.

	Materials and methods

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Materials
The Comet assay was performed using a commercially available kit (Trevigen, Gaithersburg, MD).

Comet assay
Human myeloid leukemia K562 cells were diluted with RPMI 1640 + 10% fetal bovine serum medium to a final concentration of 175 000 cells/ml. Using a 12-well plate, the following treatments were performed in a total volume of 2 ml: control cells in 0.4% methanol; 1 mM oxazepam in 0.4% methanol; 1 mM sodium phenobarbital in 0.4% methanol; 0.5 mM Wyeth 14,643 in 0.4% methanol; 100 µM hydrogen peroxide (positive control). Plates were incubated at 37°C for 3 h. Each sample (50 µl) was harvested and immediately suspended in 450 µl of 0.5% low melting point agarose in 1x phosphate-buffered saline (PBS). An aliquot of this suspension (50 µl) was then spread onto one of two rings on commercially available Comet slides (Trevigen). The agarose was allowed to harden at 4°C for 10 min. Slides were then placed in chilled (4°C) lysis buffer (2.5 M sodium chloride, 100 mM EDTA, pH 10, 10 mM Tris base, 1% sodium lauryl sarcosinate, 0.01% Triton X-100) for 30 min at 4°C and drip dried. Slides were then immersed in 50 ml alkaline buffer (250 µM EDTA, 300 mM NaOH) at room temperature for 1 h. Slides were kept in the dark during both lysis and alkaline treatments. During this incubation, cell viabilty (trypan blue method) of each sample was measured. After 1 h, slides were rinsed twice by placing in 50 ml of 1x TBE for 5 min. The slides were then electrophoresed in 1x TBE for 10 min at 17 V. Thereafter, DNA was fixed onto the slide by placing it in ice-cold methanol for 5 min, followed by ice-cold ethanol treatment for 5 min. Slides were allowed to dry overnight. Each slide was stained with 25 µl SYBR green dye (Trevigen; diluted 10-fold in PBS before use) and then viewed under a UV microscope (Nikon Microphot FXA, Hamamatsu high resolution 512 lines, Image I AT software, FITC 3 filter).

Experiments determining the effects of phenobarbital in conjunction with S9 utilized a mixture containing 0.1 M NADP, 1 M glucose 6-phosphate and 33 mM KCl in 200 mM phosphate buffer added to an aliquot of S9 from Aroclor 1254-pretreated rats so that the final protein concentration was 10 mg/ml. The mixture was sterilized by sequential filtration through 0.45 and 0.22 µm filters. The S9 mixture (1 mg/ml) was added to K562 cells along with 1 mM phenobarbital and incubated for 12 h at 37°C. The treated cells were then subjected to the Comet assay as described above.

The extent of DNA damage was determined in two ways. The first relied on calculating the comet moment, which is the integrated density in the comet tail multiplied by the distance from the center of the nucleus to the center of mass of the tail. Computations scored 25 cells for each experiment and utilized a macro available from Herbert M. Geller at http://www2.umdnj.edu/~geller/lab/comet-Scoring-Macro.txt. Because the rapidly changing intensities of the individual cells are difficult to control (Lovell et al., 1999), a large variance within each experiment is an unavoidable consequence of using this method. Therefore, a normalizing and variance-stabilizing logarithmic transformation was then applied to the calculated tail movements. After the data were transformed, analysis of variance (ANOVA) with multiple comparisons was applied with respect to the different treatment groups. In order to maintain an overall significance level of 0.05, the Tukey–Kramer adjustment was used. All analyses were performed using the SAS System statistical software package.

The second method for measuring the extent of DNA damage involved the visual scoring of individual cells according to a scheme suggested by the manufaturer (Trevigen) and used by others (Visvardis, et al., 1997; Speit and Hartmann, 1999). Briefly, cells were scored having the following characteristics: 1, intact nucleus, smooth outer edges; 2, intact nucleus, small amount of tailing; 3, intact nucleus, large amount of tailing; 4, small nucleus, large amount of tailing. Categories 1 and 2 were combined to give the percentage of cells with very little damage and catgories 3 and 4 to give those that had obvious and extensive DNA damage. At least 25 cells were scored.

Cell viability for the experiments presented in Figures 1 and 2 were determined by the trypan exclusion assay.

View larger version (26K): [in this window] [in a new window]   Fig. 1. . Comet assay for the detection of DNA damage produced by oxazepam, phenobarbital and Wyeth 14,643. Human K562 cells were exposed for 3 h to hydrogen peroxide (100 µM), oxazepam (1 mM), phenobarbital (1 mM) or Wyeth 14,643 (0.5 mM). The Comet assay was performed as described in Materials and methods. Adjusted tail moments and cell viabilities for each experiment are given in Table IA. (A) Untreated cells; (B) hydrogen peroxide-treated cells; (C) phenobarbital-treated cells; (D) oxazepam-treated cells; (E) Wyeth 14,643-treated cells. All samples were significantly different (adjusted P < 0.005) from untreated cells, except for phenobarbital-treated cells (P = 0.83).

 

View larger version (57K): [in this window] [in a new window]   Fig. 2. . Comet assay for the detection of DNA damage produced by phenobarbital in the presence of S9. The adjusted tail moments and cell viabilities for each experiment are presented in Table IB. (A) Untreated cells; (B) phenobarbital-treated cells; (C) S9-treated cells; (D) cells treated with S9 and phenobarbital.

 
The results presented here are representative of four independent Comet assays measuring the effects of oxazepam, phenobarbital and Wyeth 14,643 and four additional experiments examining the combined effects of phenobarbital and S9.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Oxazepam and phenobarbital have been found to cause mutations in DNA as revealed by the Big Blue^® transgenic mouse model (Shane et al., 1999b, 2000). Studies on Wyeth 14,643 indicate that it is also mutagenic, causing predominantly GT and GC transversion mutations (Shane et al., 1999a). As a result of our previous findings in vivo, we wanted to determine which of these DNA lesions was mediating the DNA mutations we observed. To address the effect of these compounds on cellular DNA, we exposed human cells grown in culture to them and analyzed their effects using the Comet assay. This test measures the formation of alkali-labile sites in the form of single- and double-strand breaks and the presence of baseless sites in DNA. When tests were performed on human myeloid leukemia K562 cells, oxazepam and Wyeth 14,643 were found to produce extensive DNA damage (Figure 1 and Table IA). Comparing the tail moments between these two compounds showed that oxazepam (Figure 1B) and Wyeth 14,643 (Figure 1D) produced significant amounts of DNA damage when compared with untreated cells (Figure 1A), but less than that found for exposure to hydrogen peroxide (100 µM). The amounts of DNA damage produced by oxazepam and Wyeth 14,643 were similar and not statistically different from one another. On the other hand, numerous attempts to show DNA damage by sodium phenobarbital (Figure 1C) were negative.

View this table: [in this window] [in a new window]   Table I. . Results of Comet assays with K562 cells after treatment with phenobarbital, oxezepam, Wyeth 14,643 and S9

 
Phenobarbital is known to induce certain detoxifying agents in the liver. We therefore questioned what effect an S9 liver extract might have on the ability of phenobarbital to ultimately produce DNA damage that is dependant on S9 activation. Surprisingly, as seen in Figure 2 and Table IB, the presence of S9 results in extensive DNA damage when combined with phenobarbital. The magnitude of DNA damage for each of the compounds tested, and their calculated tail moments, are summarized in Table IA and B. It is unclear why S9 on its own caused DNA damage. Nevertheless, the combination of phenobarbital and S9 produces significantly (P < 0.05) greater DNA damage than S9 alone.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
The mechanism by which oxazepam, phenobarbital or Wyeth 14,643 cause tumorigenesis in rodents is largely unknown. As measured by the sensitive ³²P-post-labeling technique, phenobarbital does not form bulky DNA adducts in Sprague–Dawley rats (Randerath et al., 1992) or in B6C3F1 mice (Whysner et al., 1998). Previous studies have concluded that oxazepam and phenobarbital are weak mutagens. Numerous theories have been proposed to explain this apparent anomaly between the mutagenic and carcinogenic properties of these compounds. These include, amongst others, the production of oxygen radicals resulting in oxidative stress (Halliwell and Aruoma, 1991) and an increased rate of cell division (Smith et al., 1991; Counts et al., 1996) which could lower the fidelity of DNA repair enzymes (Cohen and Ellwein, 1990, 1991).

Our own data shows that oxazepam, phenobarbital and Wyeth 14,643 produce G:CT:A and G:CC:G mutations (Shane et al., 1999a,b, 2000) that could clearly arise through oxidative stress. This would include the formation of DNA adducts in the form of 8-oxoguanine, which if left unrepaired can pair with thymine residues during DNA replication (Cabrera et al., 1988). Another common DNA lesion formed by oxidative stress is an apurininic/apyrimidinic (AP) site, at which DNA replication opposite the lesion generally results in incorporation of an adenine residue (Sagher and Strauss, 1983). Both of these events would explain the higher percentage of G:CT:A transversions seen in liver cells of mice exposed subchronically to the above three compounds.

Here we show that oxazepam and Wyeth 14,643 cause extensive DNA damage as revealed by the Comet assay under alkaline conditions. The types of DNA lesions that can be produced under the assay conditions employed would be a combination of AP sites and single- and double-strand DNA breaks. As noted before, the formation of an AP site could result in incorporation of an adenine residue regardless of the identity of the missing base. It is possible that single-strand breaks result in termini other than 5'-P and 3'-OH, which can be directly sealed by a DNA ligase. Indeed, formation of a 3'-P terminus could lead to an increase in error-prone DNA repair since multiple steps are required to remove the 3'-phosphate.

Although a recent study using the Comet assay reported that damage was detected in DNA of hepatocytes from mice exposed to phenobarbital (Sasaki et al., 1997), the authors were unable to exclude the possibility that the effects they detected were due to toxicity brought on by exposure to phenobarbital. In the study presented here conditions were utilized that resulted in virtually no loss in cell viability (~90% cell viability) and we found no evidence of DNA damage caused by phenobarbital. Importantly, when phenobarbital exposure was combined with an S9 liver extract, extensive DNA damage was observed in human cells. This is intriguing since phenobarbital is known to induce distinct cytochrome P-450 isozymes that mediate detoxification reactions. One such cytochrome P-450 family induced by phenobarbital is CYP2B4, which, among a variety of activities, is able to catalyze epoxidation (Buckpitt et al., 1995). It will be of interest if future studies show that the mutagenic properties of phenobarbital rely on cytochrome P-450 metabolism and, if so, if it involves formation of an epoxide.

In summary, the three compounds tested in this study produce DNA damage that could lead to mutations. Oxazepam and Wyeth 14,643 appear to produce similar types of DNA damage, whereas phenobarbital appears to be different in that it causes DNA damage via a cytochrome P-450-dependant mechanism.

	Acknowledgments

 
The authors wish to thank Dr Michael Cunningham, National Institutes for Environmental Health Sciences, for supplying the compounds used in this study.

	Notes

 
¹ To whom correspondence should be addressed. Tel: +1 225 763 0937; Fax: +1 225 763 3030; Email: deutscwa{at}pbrc.edu

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Received on December 13, 2000; accepted on May 2, 2001.

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