Mutagenesis, Vol. 14, No. 5, 479-482,
September 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Induction of genetic damage in human lymphocytes and mutations in Salmonella by trihalomethanes: role of red blood cells and GSTT1-1 polymorphism
1 Environmental Carcinogenesis Division (MD-68) and 2 Experimental Toxicology Division (MD-74), US Environmental Protection Agency, Research Triangle Park, NC 27711, USA
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Abstract |
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The brominated trihalomethanes (THMs) are mutagenic and carcinogenic disinfection by-products frequently found in chlorinated drinking water. They can be activated to mutagens by the product of the glutathione S-transferase-
(GSTT1-1) gene in Salmonella RSJ100, which has been transfected with this gene. To evaluate this phenomenon in humans, we have examined the genotoxicity of a brominated THM, bromoform (BF), using the Comet assay in human whole blood cultures exposed in vitro. No differences were found in the comet tail length between cultures from GSTT1-1+ versus GSTT1-1– individuals (1.67 ± 0.40 and 0.74 ± 0.54 µm/mM, respectively, P = 0.28). The high variability was due to the relatively weak induction of comets by BF. Combining the data from both genotypic groups, the genotoxic potency of BF was 1.20 ± 0.34 µm/mM (P = 0.003). GSTT1-1 is expressed in red blood cells but not in the target cells (lymphocytes), and expression within the target cell (as in Salmonella RSJ100) may be necessary for enhanced mutagenesis in GSTT1-1+ relative to GSTT1-1– cultures. To examine this, we exposed Salmonella RSJ100 and a control strain not expressing the gene (TPT100) to the most mutagenic brominated THM detected in Salmonella, dibromochloromethane (DBCM), either in the presence or absence of S9 or red blood cells from GSTT1-1+ or GSTT1-1– individuals. S9 did not activate DBCM in the non-expressing strain TPT100, and it did not affect the ability of the expressing strain RSJ100 to activate DBCM. As with S9, red cells from either genotypic group were unable to activate DBCM in TPT100. However, red cells (whole or lysed) from both genotypic groups completely repressed the ability of the expressing strain RSJ100 to activate DBCM to a mutagen. Such results suggest a model in which exposure to brominated THMs may pose an excess genotoxic risk in GSTT1-1+ individuals to those organs and tissues that both express this gene and come into direct contact with the brominated THM, such as the colon. In contrast, those organs to which brominated THMs would be transported via the blood might be protected by erythrocytes. Such a proposal is reasonably consistent with the organ specificity of drinking water-associated cancer in humans, which shows slightly elevated risks for cancer of the colon and bladder but not of the liver. |
Introduction |
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Bromoform (BF) and dibromochloromethane (DBCM) are brominated trihalomethanes (THMs) that are frequently found as disinfection by-products in chlorinated drinking water (International Agency for Research on Cancer, 1991
). The brominated THMs are carcinogenic in rodents at high doses, causing colon, kidney and liver tumors, and they are mutagenic in a variety of systems (Marimoto and Koizumi, 1983; International Agency for Research on Cancer, 1991). Epidemiological studies have demonstrated slightly elevated risks for cancer of the bladder and colon as well as for developmental problems among people who drink chlorinated water versus those who do not (International Agency for Research on Cancer, 1991; Morris et al., 1992; Reif et al., 1996; Koivusalo and Vartiainen, 1997; Hildesheim et al., 1998; Waller et al., 1998).
The metabolism of the brominated THMs is not completely understood. The primary route of metabolism may involve an oxidation reaction that leads to production of the dihalocarbonyl and CO2 (International Agency for Research on Cancer, 1991). However, mono- and dihalogenated ethanes or methanes, such as dichloromethane, are chemically similar to THMs and are known to undergo an oxidation reaction that results in the formation of formaldehyde. Glutathione S-transferase- (GSTT1-1) mediates this activation step in which the reduced glutathione is not consumed (Ahmed et al., 1980; Abdel-Rahman et al., 1984; Andersen et al., 1987; Thier et al., 1993, 1996; Graves et al., 1994; Hallier et al., 1994; Graves and Green, 1996). The brominated THMs likely share some of the same pathways because recent studies have shown that BF and DBCM are activated to mutagens in a transgenic strain of Salmonella (RSJ100) containing the rat homolog of the GSTT1-1 gene (DeMarini et al., 1997; Pegram et al., 1997).
GSTT1-1 is polymorphic in humans, with 20–25% of Caucasians and 50% of Asians having a homozygous deletion of this gene, resulting in a null genotype (GSTT1-1–) (Pemble et al., 1994; Kelsey et al., 1995; Wiencke et al., 1995). The results in Salmonella suggest that people carrying at least one copy of the gene (GSTT1-1+) might be more susceptible to the genotoxic effects of the brominated THMs than those missing the gene (GSTT1-1–). In a previous study, we tried to verify this hypothesis by evaluating the ability of BF to induce sister chromatid exchanges (SCEs) in vitro in whole blood cultures from the two groups of people (Landi et al., 1999). Although we found no enhanced induction of SCEs among the GSTT1-1+ individuals, SCEs may not have been a suitable end-point and/or the experimental design may have prevented detection of increased mutagenicity in GSTT1-1+ individuals because the enzyme is expressed in red blood cells, which were present in the cultures, but the enzyme is not expressed in the target cells (lymphocytes).
In the present study we have again evaluated the genotoxicity of BF in whole blood cultures from GSTT1-1+ and GSTT1-1– individuals, but we have used the single cell gel electrophoresis (SCGE or Comet) assay to score for a different end-point, DNA breaks or alkali-labile sites. A previous study (Thier et al., 1993) exposed liquid suspension cultures of Salmonella RSJ100 to dihalomethanes and showed that the glutathione S-transferase-mediated metabolites had to be generated inside the cell in order to be mutagenic. Therefore, we have examined this issue relative to THMs by exposing Salmonella RSJ100 to vapor of the most mutagenic brominated THM detected in Salmonella (DeMarini et al., 1997), DBCM, either in the presence or absence of rat liver S9 or red blood cells from GSTT1-1+ or GSTT1-1– individuals. The inability of rat liver S9 or red blood cells from GSTT1-1+ individuals to activate DBCM to a mutagen would indicate that, as with dihalomethanes, the THMs must be activated via GSTT1-1 within the target cell.
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Materials and methods |
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Blood donors and preparation
Blood was collected from four anonymous individuals after they had completed a questionnaire regarding their medical history and signed a consent form. Blood was collected by venipuncture into heparinized tubes and kept at room temperature for 1–4 h until use. For whole blood suspensions, 0.6 ml of heparinized blood was added to 9.4 ml of RPMI 1640 medium with glutamax (Gibco BRL, Rockville, MD). To obtain isolated red cells, whole blood was centrifuged in a Ficoll Isopaque (Sigma, St Louis, MO) density gradient according to the manufacturer's instructions. The red cell pellet was washed twice in RPMI 1640 medium with glutamax, concentrated by centrifugation, counted, and diluted appropriately in RPMI 1640 with glutamax. Red cells were lysed by adding an equal volume of 0.075 M KCl to the cell pellet (1010 cells/ml) and mixing vigorously using a vortex mixer. The lysed cells were then diluted in RPMI 1640 with glutamax to the appropriate concentrations.
Comet assay
BF (purity >99%; Aldrich, Milwaukee, WI) was diluted in dimethyl sulfoxide (DMSO; Burdick and Jackson, Muskegon, MI) and 50 µl aliquots of various concentrations were added to 10 ml whole blood cultures prepared as described above in glass tubes; control cultures received 50 µl of DMSO. After incubating on a roller wheel for 3 h at 37°C, the cells were prepared for the Comet assay according to standard procedures (Singh et al., 1988). Briefly, 0.5% normal melting point agarose was prepared in calcium/magnesium-free phosphate-buffered saline and used to make the first layer on the slides; 0.7% low melting point agarose prepared similarly together with 3x105 cells was used to make the second layer. After cells were lysed with lysis solution (1% sodium sarcosinate, 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl, 1% Triton X-100, and 10% DMSO, pH 10), the slides were placed for 20 min in a horizontal electrophoresis unit containing buffer composed of 1 mM Na2EDTA and 300 mM NaOH, pH 13. Cells were then electrophoresed for 30 min at 25 V and 300 mA, after which the cells were neutralized with 0.4 M Tris–HCl (pH 7.5), dried in ethanol and stored in the dark at room temperature. After staining with ethidium bromide, the cells were assessed for DNA breaks by fluorescence microscopy by measuring the lengths of the comet tails with an ocular micrometer; 100 nuclei were scored per dose, each in duplicate.
Salmonella assay
Salmonella typhimurium strains RSJ100 and TPT100 were kindly provided by Dr F.P. Guengerich (Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN). Strain RSJ100 was constructed by transfecting the base substitution strain TA1535 (hisG46, rfa, 
uvrB) with the rat glutathione S-transferase gene GSTT1-1; strain TPT100 is identical to RSJ100 except that the GSTT1-1 gene is in the opposite orientation, making it non-functional (Thier et al., 1993). The standard plate-incorporation assay was used with modifications for testing volatile chemicals (Hughes et al., 1987) as described previously (Pegram et al., 1997). S9 was from aroclor 1254-induced Sprague–Dawley rat liver.
Briefly, DBCM vapor (Pegram et al., 1997) was injected into sealed 4 l Tedlar bags containing: (i) a volume of sterile air necessary to achieve the desired final chemical concentration; (ii) six glass Petri dishes in which the bacteria and red blood cells (whole or lysed, prepared as described above) or Sprague–Dawley aroclor 1254-induced rat liver S9 had been plated just prior to exposure. The six plates/bag represented one plate of each S9 or red cell concentration. A duplicate set of six plates was placed in a second bag and these bags were exposed in parallel. Thus, the experiment was performed once, but duplicate sets of plates (each set in separate bags) were used to generate replicated data. A Hewlett Packard 5890A gas chromatograph equipped as described previously (Pegram et al., 1997) was used to quantify the vapor concentrations in the bags. After 24 h of exposure to DBCM vapor at 37°C, the plates were removed from the bags, incubated for an additional 48 h at 37°C, and the revertant (rev) colonies counted.
Genotyping
A multiplex PCR was performed as described previously except that 6.2 instead of 3.3 mM MgCl2 was the final concentration in the reaction (Bell and Pittman, 1998). Briefly, genomic DNA was isolated by standard methods from a portion of the blood samples and a multiplex PCR was performed in which GSTM1-1 and GSTT1-1 were co-amplified together with ß-globin as an internal positive control. Although GSTM1 is not known to be involved in THM metabolism, our laboratory performs the multiplex genotype assay routinely. Consequently, data regarding a possible influence of the GSTM1 gene on THM metabolism were also available to us for consideration.
Statistical analysis
The genotoxic potencies of BF in the Comet assays were calculated initially as the slope of the dose–response curves using linear regression analysis. However, because the concentration–response relationships were generally not linear, the response was recalculated as the net increase in average comet tail lengths above the spontaneous value at the top dose. Student's t-test was used to compare the responses between the different genotypes. In the Salmonella experiments, data for each dose of DBCM and each concentration of red cells were pooled. The comparisons between genotypes or among groups of plates with or without red cells were done using Student's t-test.
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Results and discussion |
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Table I
shows the age, sex, and genotype of the subjects whose blood was used for the Comet assay. As shown previously with SCEs (Landi et al., 1999), the DNA damaging potency of BF was not significantly different between GSTT1-1+ and GSTT1-1– subjects, with the genotoxic potency being 1.67 ± 0.40 versus 0.74 ± 0.54 µm/mM, respectively (P = 0.28). Nonetheless, as with SCE induction (Landi et al., 1999), BF also induced DNA damage, as evidenced by a weak but significant increase in DNA tail length (1.20 ± 0.34 µm/mM, P = 0.003), based on combining the data from both genotypic groups (Table II). SCEs and the Comet assay are highly sensitive assays for DNA damage, and it seemed unlikely to us that if there were a different genotoxic response between the two groups of people that we would not have detected it using one or other of these end-points.
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Although BF was not more genotoxic (for either SCEs or DNA damage) in lymphocytes cultured in the presence of red blood cells from GSTT1-1+ versus GSTT1-1– individuals, BF and the other brominated THMs are activated to potent mutagens by the product of the GSTT1-1 gene when the gene is expressed inside the target cells (Salmonella RSJ100) (DeMarini et al., 1997
; Pegram et al., 1997). A previous study with dihalomethanes in Salmonella showed that activation by GSTT1-1 outside the cell would not produce any mutagenesis (Thier et al., 1993). Thus, instead of the end-points (SCEs and DNA damage) being inappropriate, we suspected that the problem in detecting increased genotoxicity in the presence of GSTT1-1 may have been due to the fact that the target cells (lymphocytes) did not express GSTT1-1, which was expressed, instead, by the co-cultured red cells. Therefore, to explore the effect of the presence of red cells in the culture, we performed the following studies in Salmonella. Salmonella cells were exposed to DBCM in the presence of whole or lysed red cells from donors who were either GSTT1-1+ or GSTT1-1–. This permitted us to evaluate the influence of extracellular GSTT1-1 (supplied by red cells from GSTT1-1+ donors) on the mutagenicity of DBCM in strains of Salmonella that can (RSJ100) or cannot (TPT100) activate DBCM to a mutagen.
Red cells from either genotypic group were not able to activate DBCM in the non-expressing strain TPT100 (Table III). However, red cells from both genotypic groups completely repressed or abrogated the ability of the expressing strain RSJ100 to activate DBCM to a mutagen (Table III). This effect was observed irrespective of the genotype through a range of concentrations of either whole or lysed red cells. Only the highest concentrations of whole or lysed cells (107–109/plate) appeared to be toxic to Salmonella, as evidenced by an ~50% reduction in the spontaneous mutant yield relative to plates with low concentrations of red cells or no red cells (Table III).
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The fact that red cells from even GSTT1-1– individuals could totally inhibit the mutagenicity of DBCM in strain RSJ100 indicated that erythrocytes can sequester or inactivate DBCM, preventing the chemical from reaching the Salmonella cells and that GSTT1-1 does not exert any role in this mechanism. Indeed, the efficient interference exhibited by red cells in this study in Salmonella may explain the relatively low frequencies of BF-induced SCEs in lymphocytes in the presence of red cells (Landi et al., 1999
).
To further examine this inhibition of DCMB mutagenicity by red cells, we replaced red cells with rat liver S9. The results showed that rat liver S9 was not able to activate DBCM in the non-expressing strain TPT100 and that S9 had no effect on the ability of the expressing strain RSJ100 to activate DBCM (Table IV). Thus, even the presence of S9 protein did not result in any significant non-specific binding of the DBCM molecule to significantly reduce the mutagenic effect of this THM in RSJ100.
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Considering these results and other data (DeMarini et al., 1997
; Pegram et al., 1997; Landi et al., 1999), THMs are clearly activated to mutagens in exposed cells that express GSTT1-1 (Salmonella RSJ100), but human erythrocytes from either GSTT1-1+ or GSTT1-1– donors appear to sequester and/or inactivate THMs, preventing the mutagenicity of THMs in cells that either express GSTT1-1 (Salmonella RSJ100) or do not express this enzyme (Salmonella TPT100 and human lymphocytes).
Such results suggest a model in which exposure to THMs (e.g. from chlorinated drinking water) may pose an excess genotoxic risk in GSTT1-1+ individuals to those organs and tissues that both express this gene and come into direct contact with the THM. This could include the gastrointestinal tract, such as the colon (Juronen et al., 1996). In contrast, organs to which THMs would be transported via the blood, such as the liver, might be considered to be protected by erythrocytes. This proposal is in reasonable agreement with the organ specificity of drinking water-associated cancer in humans, which shows slightly elevated risks for cancer of the rectum and bladder but not of the liver (International Agency for Research on Cancer, 1991; Morris et al., 1992). Although some portion of ingested THMs would contact the bladder after filtration through the blood (and contact with red cells), it may be possible that another portion may arrive at the bladder without prior contact with or binding to red cells. In this form, THMs could then enter bladder cells and be activated in situ. GSTT1-1– people might be at less risk than GSTT1-1+ individuals; however, THMs may be activated by other pathways, presenting a risk regardless of GSTT1-1 status (DeMarini et al., 1997).
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Acknowledgments |
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We thank the staff at Integrated Laboratory Systems for providing the training and facilities to perform the Comet assay. S.L. acknowledges the support of a Research Associateship Award from the National Research Council, US National Academy of Sciences. This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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Notes |
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3 To whom correspondence should be addressed. Tel: +1 919 541 1309; Fax: +1 919 541 0694; Email: landi.stefano{at}epa.gov
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