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Cancer Epidemiology Biomarkers & Prevention Vol. 8, 609-614, July 1999
© 1999 American Association for Cancer Research

Analysis of DNA Strand Breaks, Oxidized Bases, and Glutathione S-Transferase P1 in Human Colon Cells from Biopsies1

Beatrice L. Pool-Zobel2, Salomon L. Abrahamse, Andrew R. Collins, Wolfgang Kark, Roland Gugler, Daniela Oberreuther, Eberhard Günther Siegel, Sylvia Treptow-van Lishaut and Gerhard Rechkemmer

Institute of Nutritional Physiology, Federal Center for Nutrition Research, Karlsruhe, Germany 76131 [B. L. P-Z., S. L. A., D. O., S. T-v. L., G. R.]; Rowett Research Institute, Aberdeen, Scotland, United Kingdom AB21 9SB [A. R. C.]; Städtisches Klinikum, Karlsruhe, Germany 76131 [W. K.]; and St. Vinzentius Krankenhaus, Karlsruhe, Germany 76131 [E. G. S.]


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The balance of genetic damage and deactivating enzymes is decisive for cancer risk. To assess these factors in normal human colon cells, we determined background levels of DNA breaks or oxidized bases and of glutathione S-transferases (GSTs) as potential biomarkers of risk and chemoprevention, respectively. Also, genotoxicity by compounds involved in lipid peroxidation was determined to elucidate possible sources of damage. Cells were isolated from sigmoid biopsies of 51 donors and processed with the comet assay to reveal genetic damage. GST proteins were analyzed immunologically. HT29 clone 19A colon tumor cells, resembling primary cells, were treated with 2-trans-hexenal (400 µM) or hydrogen peroxide (75 µM) and processed for damage. Fifteen percent of primary colon cells contained strand breaks; 22% contained additional oxidized bases, with distinct sex differences. Similar damage was found in HT29 clone cells and is induced by both test compounds. GST levels were similar in both cell types. The comet assay is sufficiently sensitive to detect oxidative genetic damage in small amounts of cells from small amounts of biopsies. Lipid peroxidation is a possible risk factor. Together with GST as a potential biomarker of chemoprevention, the technique may serve as a valuable biomarker to assess exposure to risk factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Continuous exposure of the colon epithelium to genotoxic agents is expected to cause DNA damage in the cells (1 , 2) . Additionally, within mammalian cells, oxidative stress, resulting from a prevalence of oxidants, produces specific types of cellular damage to macromolecules (3 , 4) . The damage by oxidants and by other genotoxic agents is expected to play a role in the development and generation of cancer (5) . The extent of damage is dependent on detoxifying enzymes such as GSTs,3 antioxidant defense systems, macromolecules that scavenge electrophilic intermediates, and DNA repair systems (6) . In the colon, the special exposure situation reflecting the antioxidant and pro-oxidant status of the digesta needs additional consideration (7) . Numerous biochemical and analytical methods have been developed to assess oxidized macromolecules in the human body, especially in body fluids. For elucidating processes of carcinogenesis, the detection of oxidative DNA bases and other genetic damage within tumor target tissues, however, is of primary interest. Usually, the detection of oxidized DNA lesions (8-hydroxyguanine or 8-hydroxydeoxyguanosine) involves the isolation of DNA from tissues with subsequent analysis by gas chromatography, HPLC, or 32P-DNA labeling and two-dimensional TLC (8 , 9) . Immunohistochemical techniques are also available (10) . Generally, these assays cannot be applied to the small quantities of tissue available from biopsies of individual donors owing to limits of detection levels. Therefore, only a few reports on oxidative and other DNA damage in the colon are available. Examples are studies by Oliva et al. (11) who recently reported evidence of higher levels of 8-hydroxyguanosine and other markers of oxidative stress and genetic damage in colon tumor tissue versus normal mucosa for several patients. Erhard et al. (12) have subsequently shown that diets rich in fat and poor in dietary fiber increase the in vitro formation of reactive oxygen species in the human feces. Thus, evidence is just beginning to point out the mechanisms by which dietary factors may contribute to enhancing molecular events that lead to colon cancer. Obviously, more research is necessary to obtain knowledge on the basal levels of oxidative DNA damage in human colon cells and, subsequently, to elucidate which physiological factors may contribute to inducing oxidative stress and genetic damage in this tissue.

In this context, we have recently further developed the technique of single-cell gel electrophoresis ("comet" assay) to study DNA damage in cells from human biopsies (13) . We have examined the effects on colonocytes of several genotoxic carcinogens that are postulated to be risk factors of colon carcinogenesis (2) . In the present study, we have determined the basal levels of DNA damage by processing the cells directly after isolation, without in vitro incubation. Moreover, we have introduced a modification of the assay by incubating the slides after lysis with endonuclease III to reveal specific oxidative DNA damage (14 , 15) . The endogenous levels were assessed in primary human colon cells from biopsies as well as in cells of a human intestinal tumor cell line (HT29) and in cells of its differentiated clone (HT29 clone 19A), which has features more closely representing primary cells than the parent cells (16) .

Endogenous genetic damage is expected to be a result of reactions of the DNA with oxygen-free radicals and with products arising from the peroxidation of polyunsaturated fatty acids. These reactive compounds are formed during cell metabolism, signal transduction, or indirectly by various types of toxicants (17) . We investigated H2O2, one of the most frequent peroxides that decomposes in the presence of transition metals to the DNA-damaging hydroxyl radical (18) . We also investigated 2-trans-hexenal, one of the reactive aldehydes formed from the complex peroxidation of the fatty acids (19) . The compounds were investigated in HT29 clone 19A human colon tumor cells, which resemble primary cells in respect to endogenous levels of genetic damage, to determine whether these stress factors can actually be contributors to the observed endogenous damage in colon cells.

Finally, GST subunit P1, an important component of the chemoprevention system, was quantitatively determined by reversed-phase HPLC and by ELISA. The quantitative presence of this enzyme system is important for defining the sensitivity of cells toward genotoxic factors, and it may be induced by dietary factors (20) . Therefore, the work outlined here was additionally aimed at developing the determination of this detoxifying enzyme as a biomarker of susceptibility or as a biomarker of chemoprevention in human colon cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and Reagents.
Hydrogen peroxide (Cas 7722-84-1) was obtained as a 30% aqueous solution from Merck (Darmstadt, Germany). 2-trans-hexenal (Cas 6728-26-3) >= 99% pure was from Sigma (Deisenhofen, Germany). Protease and collagenase were from Amresco (Solon, OH) and Boehringer (Mannheim, Germany), respectively. DMEM was from Life Technologies, Inc. (Eggenstein, Germany). Kits and antibodies to determine GST proteins were from Biotrin (Sinsheim-Reihen, Germany). All of the other chemicals were of analytical grade or complied with the standards needed for tissue culture experiments.

Donors of the Colon Biopsies and Isolation of Cells.
The biopsy donors were submitted to the hospitals for various reasons and were subjected to diagnostic colonoscopy to exclude either polyps or colorectal carcinoma after occult fecal blood test had been positive. The excision of biopsies was performed after the patients gave their informed consent. The ethical committee of the Landesärztekammer Baden Württemberg approved the study. Here, samples were only evaluated from patients for whom the findings were negative with respect to benign or malignant tumors or inflammatory bowel disease. Six biopsies were obtained from macroscopically healthy colon sigmoid tissue and transported on ice to the laboratory within 1–2 h. The biopsies were minced with fine scissors and incubated with 6 mg of proteinase K and 3 mg of collagenase in 3 ml of HBSS for 30 min in a shaking water bath at 37°C (2) . The suspensions were then diluted with HBSS to a volume of 15 ml and centrifuged for 6 min at 139 x g. Pellets were resuspended in 6 ml of HBSS for further processing. Viability of cells was determined by trypan blue exclusion (13) .

Human Tumor Cell Lines.
The human colon cell line HT29 was established by J. Fogh (Memorial Sloan Kettering Cancer Center, New York) in 1964 (quoted in Ref. 16 ). Passages 30–48 were used in this study. The subclone 19A was terminally differentiated with 5 mM sodium butyrate and characterized by Augeron and Laboisse (21) . Passages between 8 and 16 and 18–22 for the experiments with H2O2 and 2-trans-hexenal, respectively, were available for this study.

Tissue Culture.
Cells were maintained in stocks at -80°C, thawed, and grown in tissue-culture flasks with DMEM supplemented with 10% FCS and 1% penicillin/streptomycin. This culture containing 45–60 x 106 cells was trypsinized and subcultivated at a dilution of 1:8 in T75 flasks with supplemented DMEM. Two medium changes occurred on days 2 and 5 with 16 ml of DMEM. On day 6, the confluent cell layer was trypsinized with 1–1.5 ml of trypsin/versene (1:10 v/v) for maximal 10 min. The cells were gently shaken off the plastic flask and resuspended at appropriate concentrations (2 x 106 cells/ml) in cold DMEM. The viability of cells was determined by trypan blue exclusion. This protocol was strictly adhered to for both genotoxicity determination and for the detection of GST, to exclude differences in cell properties owing to culture conditions (6 , 22) .

Determination of Genetic Damage.
Ten µl of the cell suspensions (containing 2 x 105 HT29 cells or 3–4 x 105 primary human colon cells) were mixed with 75 µl of 0.7% low-melting-point agarose and distributed onto microscope slides coated with 0.5%-normal-melting agarose. After the solidification of the agarose, slides were covered with another 75 µl of 0.7% low-melting-point agarose and then submersed into a lysis solution [100 mM Na2EDTA, 1% Triton X-100, 2.5 M NaCl and 1% N-lauroylsarcosin sodium salt, 10% DMSO, 10 mM Tris (pH 10) for at least 60 min]. Slides being processed for OxBs were washed with endonuclease III buffer [40 mM HEPES-KOH, 0.1 M KCl, 0.5 mM Na2EDTA, 0.2 mg/ml BSA fraction V (pH 8; 3) for 5 min] and incubated with endonuclease III in buffer (1 µg/ml, 50 µl/slide), sealed with a coverslip for 45 min at 37°C. All of the slides were placed in an electrophoresis chamber containing alkaline buffer [1 mM Na2EDTA, 300 mM NaOH (pH 13)] for DNA unwinding. After 20 min, the current was switched on and electrophoresis was carried out at 25 V, 300 mA for 20 min. The slides were removed from the alkaline buffer and washed three times for 5 min each time with neutralization buffer [0.4 M Tris (pH 7.5)]. Slides were stained with ethidium bromide (20 µg/ml; 100 µl/slide). All of the steps beginning with the isolated cells were conducted under red light. For each human donor, one slide was processed with and one without endonuclease III, for the determination of OxBs and DNA Sbs, respectively. The tests with HT29 cells and HT29 clone 19A cells were performed in triplicate, and the experiments were independently reproduced at least four times.

Treatment of Cells with Chemicals.
2-trans-hexenal was dissolved in 10% DMSO in 0.9% NaCl. H2O2 was dissolved in 0.9% NaCl. Up to 10 µl of these solutions or of the solvents were added to 1 ml of cell suspension containing 2 x 106 HT29 clone cells. The suspensions were incubated for 30 min in a shaking water bath at 37°C and centrifuged. The pellet was taken up in agarose, distributed onto slides, and then processed according to the protocol for determining genetic damage as described above.

Each compound was assessed in triplicate per determination. Four independent reproductions were performed (Table 3)Citation .


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  		Table 3 Induction of DNA damage in HT29 clone 19A cells by H2O2 and 2-trans-hexenal

 
Evaluation of the Comet Assay.
Microscopical evaluation of the images was quantified using the image analysis system of Perceptive Instruments (Halstead, United Kingdom). Fifty images were evaluated per slide, and the %TI was scored. The "tail intensity yield" (values of slides treated with endonuclease III minus values from corresponding slides without the enzyme) is a reflection of the levels of oxidative DNA damage.

In some cases, the extent of damage is presented as the "% of comets" in a population. A comet is defined as an image with a value exceeding 6%TI.

Determination of Protein Subunits of GST.
Cell pellets containing proteins from 1.3–9.9 x 106/2.5 ml HT29 cells (for ELISA), 40–77 x 106/3 ml HT29 cells (for HPLC), and 1.3–3.7 x 106/2 ml human colon cells (for ELISA) were taken up in cell destruction buffer [20 mM Tris-HCl, 250 mM saccharose, 1 mM DTT, 1 mM PMSF, 1 mM EDTA (pH 7.4)] and homogenized by vigorous Ultra-Turrax treatments for 1 min. For the determinations with HPLC, GST proteins were captured on affinity columns containing epoxy-activated Sepharose 6B to which S-hexylglutathione is linked. The GST subunits were then eluted from the column using 100 mM Tris-HCl, 1 mM Na2H2EDTA x 2H2O, 0.2 mM DTT, 200 mM NaCl, and 2.5 mM S-hexylglutathione (pH 7.8). The concentrated eluates were then subjected to HPLC, and the GST subunits were eluted using a special CH3CN (TFA)/H2O(TFA)-gradient program on RP18. Detection was by UV absorption at 214 nm. The protein subunit P1 appeared at about 29.6 min at a pressure of about 1700 psi and a flow rate of 0.6 ml/min. The area under each peak was determined as a quantitative estimation for each of the three individual experiments. Quantitative calibration was previously performed with a standard protein from Biotrin (Sinsheim-Reihen, Germany; Nr. BIO 52 AG PI).

Because the cell quantities of the human biopsies were not sufficient for an analytical determination of GST proteins using HPLC, these and the other cell types were also analyzed by using immunologically linked enzyme assay for GST P1 (Biotrin, Sinsheim-Reihen, Germany).

Statistical Evaluation.
For data analysis, we have classified our subjects into groups of males and females, with subdivisions of each for ages >60 and >50 years old. Furthermore, 15 individuals of each sex were age-matched (Table 1)Citation .


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  		Table 1 Basic data and parameters of DNA breakage (Sbs) or oxidized DNA bases (OxBs) in human colon cells isolated from sigmoid biopsies

 
The Prism software (Graph Pad) was used for establishing two-sided significance levels. As is specified in the tables and graphs, paired and unpaired t tests or linear regression values were used whenever appropriate. When triplicate parallel determinations were available (studies with cultivated cells), the means were first calculated and taken as a basis for the analyses outlined above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Colon cells of human biopsies can be isolated in sufficient quantity and quality for in vitro toxicology studies. All together, we originally had access to the biopsies of 66 patients. Data of 15 patients were excluded because they were either tumor patients (Hodgkin’s lymphoma, bone metastases, melanoma, colonic polyps) or afflicted with other diseases (ulcerative colitis, Crohn’s disease, liver cirrhosis) considered to modulate status of endogenous genetic damage. Some samples were excluded because there was a delay that exceeded 2–3 h between biopsy excision in the clinic and work-up in the laboratory. Samples were taken from macroscopically healthy colon tissue of the remaining 51 patients, who were otherwise nonapparent in terms of clinical diagnosis or medication. Their age, weight, and height were recorded and the BMI was calculated from the latter two vital data parameters. Samples were available from 31 males (ages 24–83; median, 60 years) and 20 females (ages 35–84; median, 62 years) from two local hospitals. The age-matched groups consisted of 15 males and 15 females with a mean age of 62 years. As is shown in Table 1Citation , approximately 2.4 million cells can be obtained from the biopsies of each donor, with viabilities of over 90%. No differences in the quality of cells (yield and viability) were seen between the different groups.

Human colon cells contain measurable amounts of DNA breaks and oxidized pryrimidine bases. Fifteen percent of primary colon cells were comets in the test for Sbs (standard comet assay); 22% were comets in the test for oxidized bases (test modification with endonuclease III). As is shown in Table 1Citation , this is equivalent to a %TI of 5.9 ± 0.7 and 11.7 ± 1.1 for Sbs and additional oxidized bases, respectively. No statistically significant correlations were found with age, BMI, or sex and damage (% fluorescence in comet tail). Males have significantly more oxidized DNA bases than females.

Cells of a human intestinal tumor cell line also contained DNA breaks and additional OxBs. The endogenous levels were compared for the highly undifferentiated parent cell line, its differentiated subclone, and the primary colon tumor cells to elucidate which cell line resembles the primary cells to a greater extent in terms of genetic damage. It is apparent from Table 2Citation that the differentiated tumor cells had significantly lower levels of Sbs and oxidized bases than the primary cells. In contrast, there were no significant differences between clone cells and primary cells. Thus, the clone cells were used as the model cell line to determine to which extent lipid peroxidation-related compounds can induce the types of damage that were shown to occur endogenously in colon cells. Primary colon cells were not suitable for these studies because of the high interindividual variations of sensitivity.


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  		Table 2 Parameters of DNA breakage in HT29 human tumor colon cells

 
The results for the HT29 clone cells treated with H2O2 and 2-trans-hexenal are shown in Table 3Citation and reveal that the peroxide induces Sbs. 2-trans-hexenal also induces Sbs and, additionally, oxidized bases at the lower concentration tested (Table 3)Citation .

The HPLC analysis of GST from HT29 stem and clone 19A cells showed that GST-P1 is present in quantities of 1.86 ± 0.14 µg and 1.71 ± 0.21 µg per 106 cells (n = 3), respectively (Fig. 1)Citation , whereas other isoproteins were less abundant. The advantage of this technique is that the quantification of the protein is accurate and that it allows the simultaneous detection of several isoproteins in one sample. HPLC determination, however, could not be performed with primary human cells, because of insufficient material for this type of measurement. Instead, GST-P1 was, therefore, analyzed in primary cells using an ELISA. For comparative purposes, the determinations were also performed in the human colon tumor cell lines. The results are shown in Table 4Citation . Interestingly, approximately 40-fold higher GST-P1 protein concentrations were observed in both tumor cells lines using the HPLC technique in comparison with ELISA, which measures only immunologically reactive sites of GST-P1. Nevertheless, by using ELISA, it was at least possible to analyze the minute samples of cells from biopsies and, thus, to compare the three cell types. The results presented in Table 4Citation show that there were no significant differences in GST-P1 contents, and that the variability was especially large in the primary cells.



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  		Fig. 1. HPLC chromatograms of affinity-purified GST isoenzymes of HT29 stem cells (A) and HT29 clone 19A cells (B). HPLC chromatogram of human standards GST-A1, -A2, -M1a, -M2, and -P1 (C). The HPLC conditions were as described in "Materials and Methods." X-axis, retention time (min); Y-axis, absorbance at 214 nm. The identity of each peak is indicated.

 

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  		Table 4 GST-P1 in human colon cells (means ± SE; no significant differences were found)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Methylation, deamination, depurination, and oxidized DNA bases are expected to be the most important endogenous types of DNA damage. Estimates of total damage are in the range of approximately 100 nmol per day and per cell (5) . In human urine, the levels of oxidative DNA damage have been assessed by measuring the excretion mainly with chemical analytical techniques. These, however, tend to overestimate the damage owing to artifactual, additional oxidized DNA bases arising during the phenol extraction of DNA from the cell (8 , 23) . In contrast, the comet assay does not require this extraction step because cells are monitored directly. Accordingly, in studies with the endonuclease III modification of the assay, a 10-fold lower level of endogenous DNA damage has been found in lymphocytes than had been estimated by Ames and colleagues (14 , 24 , 25) . Our present study has shown for the first time in a large number of individuals that genetic damage and oxidative DNA damage is also detectable in nontransformed human colon cells. The values are similar to those measured in lymphocytes, which, in one of our previous studies, have been shown to contain 2.6 ± 0.3 to 4.6 ± 0.5% TI (strand breaks) or 11.5 ± 1.2 to 34.4 ± 3.8%TI (oxidized bases and Sbs), depending on diet. Lower values of genetic damage were found after the consumption of vegetable juices (26) .

The consequences of DNA damage are manifold. In addition to mutagenic lesions, blocks of transcription or replication leading to altered gene regulation and expression are probable. Therefore, the knowledge of DNA damage and its potential modulation by nutritional factors should be an intrinsic part of risk estimation. We can only roughly speculate on the origin of the endogenous damage within these cells and the consequences that may be expected. A major source of damage could be the genotoxic action of lipid peroxidation compounds, These are, however, very complex and heterogeneous (19) . Our studies here have shown that the endogenous damage could very well be due to actions of lipid peroxidation products, like H2O2 and 2-trans-hexenal. Probably other types of genotoxins occurring in the colon could be involved as well. Thus, we have also recently shown that several food contaminants and diet-related endogenous products can act genotoxicly in primary cells of human colon biopsies (2) . Additional systematic studies of this type are needed to estimate in which relative proportions individual exogenous and endogenous genotoxins contribute to the overall DNA-damaging burden within the colon and how this burden contributes to cancer induction and progression.

In any case, the degree of DNA damage is based on the genotoxic or oxidative stress prevailing in a given tissue, the levels of endogenous detoxifying systems, and the DNA repair capacity. For this reason, it is expected that different tissues will have different levels of oxidative damage and that the damaged bases that can be estimated in the urine or in human lymphocytes are not always accurate surrogates of the damage occurring in the tumor target tissues.

The colon may be especially susceptible to oxidants (and antioxidants) by the route of the gut lumen (7) . This contains bacteria that can generate free radicals, hydrogen peroxide, and genotoxins (12 , 27, 28, 29, 30, 31, 32) . Similarly, antigenotoxins may prevail to prevent damage induction (33, 34, 35) . Colon cancer may be the result of oxidants and genotoxins being more available than antigenotoxins as a result of specific dietary regimens (7 , 36) . Therefore, in addition to measuring exposure to carcinogens or genetic damage, it is also important to monitor the levels of chemopreventive systems. GST-P1 may be an important marker of this type. It is the abundant isoenzyme of the super-GST-enzyme family in the colon (37) . Moreover, as we have shown here by using ELISA, GST-P1 can be detected in the small samples derived from biopsies. It is important for colon cells because it is not only effective in deactivating a large range of genotoxic environmental carcinogens but also detoxifies physiologically occurring aldehydes and base propenals (38) . Furthermore, it is feasible to use the detection of GST-P1 protein as a biomarker of chemoprevention in dietary intervention studies because the enzyme may be induced by food ingredients (20 , 39) . Using the same detection technique, our own studies have previously shown that lymphocytes contain significantly (P < 0.01) lower levels of GST-P1 (11.7 ± 6.1 ng/106 cells, n = 6 individuals) than colon cells (Table 4)Citation . The protein levels, however, can be induced by diet (consumption of vegetable juices) to yield 24.3 ± 6 ng GST-P1/106 cells (n = 6), a value which, however, is still significantly (P < 0.05) lower than the amounts in primary colon cells shown in Table 4Citation (40) . The ELISA method used here determines only an immunreactive enzyme that may underestimate the total protein actually present. However, in contrast to the more accurate HPLC determination, it has the advantage that the small amounts of material that can be isolated from the colon biopsies suffice to perform the assay. Nevertheless, the additional measurement of enzyme activity (if this can be developed on a sufficiently small microscale basis) and the determination of mRNA are further developments that we are presently pursuing to enhance the utilization of GST-P1 as a biomarker of chemoprevention.

Significantly higher levels of oxidized DNA bases were detected in males than in females. The reasons for this are not clear. Oxidized DNA bases may be the result of compounds like peroxides and ROS (26) . It cannot be speculated in what manner males or females are differently exposed to these compounds. It is probable that hormonal influences or sex-related differences in metabolic activation or inactivation may be contributing factors. For the example of GST, it has been found that the GST-P1 isomer is expressed more in colon tissue from males than from females, and the specific activity is higher (41) . This finding is in apparent contradiction to our results because in our study, males had more oxidative damage. However, reactive oxygen species, peroxides, and other factors causing oxidative DNA damage may be detoxified by other enzyme systems (catalases, superoxide dismutases, glutathione peroxidases), for which sex-related differences in the colon are not known. Obviously, more research is needed to identify the type of oxidants occurring in the colon and how they are deactivated by different enzyme systems in this tissue and, moreover, how the factors are modulated by genetics or sex and especially by the diet.

In conclusion, this approach of using the comet assay can be developed into a very useful biomarker of risk, and the determination of GST-P1 can be used as a biomarker chemoprotection. The joint determination of the two biomarkers has already been used with human lymphocytes isolated from subjects during a dietary intervention study with vegetable juices (26 , 40) . The results of the trial have shown that in human lymphocytes genetic damage is reduced, whereas GST is induced, through the diet. A similar type of monitoring of genetic damage and of detoxifying enzymes in the target cells of colon cancer will lend additional value for understanding the impact of colon cancer chemoprevention trials. The study reported here is another step in this direction and provides unique information on the baseline values of these two parameters in human colon cells.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by the European Community, Grants AIR-CT94-0933, ERB7C7-15-CT96-1012, and FAIR-CT-95-0894. Back

2 To whom requests for reprints should be addressed, at Department of Nutritional Toxicology, Institute for Nutrition, Friedrich-Schiller-University of Jena, Dornburger Strasse 25, 07743 Jena, Germany. Phone: 49-0-3641-949-670; Fax: 49-0-3641-949-672; E-mail: b8pobe{at}uni-jena.de Back

3 The abbreviations used are: GST, glutathione S-transferase; BMI, body mass index; OxBs, oxidized pyrimidine bases; PMSF, phenylmethylsulfonyl fluoride; Sbs, strand breaks; % TI, % fluorescence in tail (tail intensity); HPLC, high-pressure liquid chromatography, TFA; trifluoroacetic acid. Back

Received 11/ 9/98; revised 4/ 5/99; accepted 3/ 1/99.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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