DNA damage and repair in human lymphocytes and gastric mucosa cells exposed to chromium and curcumin

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Teratogenesis, Carcinogenesis, and Mutagenesis 19:19–31 (1999)

DNA Damage and Repair in Human Lymphocytes and Gastric Mucosa Cells Exposed to Chromium and Curcumin Janusz B»asiak,1* Andrzej Trzeciak,1 Ewa Ma»ecka-Panas,2 Józef Drzewoski,2 Teresa Iwanienko,3 Irena Szumiel,3 and Maria Wojewódzka3 1

Department of Molecular Genetics, University of Lodz, Lodz, Poland Department of Digestive Tract Diseases and Metabolic Disturbances, Medical University of Lodz, Lodz, Poland 3 Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Warsaw, Poland 2

Human population can be considered as a subject of combined exposure to chemicals. Hexavalent chromium is a well-known mutagen and carcinogen. Curcumin, a popular spice and pigment, is reported to have antineoplastic properties. The single cell gel electrophoresis (Comet assay) is a sensitive technique that allows detecting double- and single-strand DNA breaks caused by a broad spectrum of mutagens. In the present work the ability of curcumin to reduce DNA damage induced by chromium in human lymphocytes and gastric mucosa (GM) cells was investigated by using the comet assay. Chromium at 500 mM evoked DNA damage measured as significant (P < 0.001), about a two-fold increase in comet tail moment of both lymphocytes and GM cells. Curcumin at 10, 25, and 50 mM also damaged DNA of both types of cells in a dose-dependent manner: the increase in the tail moment reached about twenty times of the control value (P < 0.001). The combined action of chromium at 500 mM and curcumin at 50 mM resulted in the significant (P < 0.001) increase in the comet tail moment of both types of cells. In each case, treated cells were able to recover within 60 min. Our study clearly demonstrates that curcumin does not inhibit DNA damaging action of hexavalent chromium in human lymphocytes and GM cells. Moreover, curcumin itself can damage DNA of these cells and the total effect of chromium and curcumin is additive. Further studies are needed to establish the role of interaction of curcumin with DNA in carcinogenesis. Teratogenesis Carcinog. Mutagen. 19:19–31, 1999. © 1999 Wiley-Liss, Inc.

Contract grant sponsor: University of Lodz; Contract grant number: 505/674; Contract grant sponsor: Medical University of Lodz; Contract grant number: 502-11-422 (119). *Correspondence to: Janusz B»asiak, Department of Molecular Genetics, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland. E-mail [email protected]

© 1999 Wiley-Liss, Inc.

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Key words:

combined action of chemicals; comet assay; DNA strand breaks; carcinogenanticarcinogen interaction; single cell gel electrophoresis

INTRODUCTION

The human environment contains a great diversity of both natural and manmade mutagens and carcinogens, as well as many natural antimutagens and anticarcinogens. Their main sources are the diet, natural and occupational environments, and life-style. Humans are continuously subjected to combined exposure to many factors, e.g., exposure to a chemical in the work place can be amplified/suppressed by a mutagen/antimutagen present in the diet. Co-influence of some factors can be casual or intentional, as for example in chemoprevention. Unfortunately, prevention strategies for cancer involving reduction or complete elimination of human exposure to diverse environmental factors may not always be possible; however the agents, which are known or suspected to alleviate carcinogenic potential of these factors, can be included in the human diet. The carcinogenic properties of hexavalent chromium [Cr(VI)] is well established and its genotoxicity has been reported in many tests [1]. Chromium is widely used in numerous industrial processes, and as a result, it is a contaminant of many environmental systems [2]. Hexavalent chromium compounds are shown to induce DNA damage in vitro [3] and in vivo [4]. These effects of hexavalent chromium are generally attributed to its cellular uptake because Cr(VI), in contrast to Cr(III), can easily pass the cell membrane by the sulfate anion system [5]. Cr(VI) is reduced inside the cell through reactive intermediates, such as Cr(V) and Cr(IV), to the more stable trivalent form by cellular reductants, including vitamins B2 and C, flavoenzymes, and glutathione [5]. This reduction process causes the generation of active oxygen species [6], which can introduce oxidative DNA damage. DNA damages evoked by Cr(VI) in cell lines were suppressed by a hydroxyl radical scavenger and by vitamin E, which decreased the level of Cr(V) [7]. Some groups of workers are occupationally exposed to chromium at elevated level. It was reported that welders exposed to this metal showed an increased level of single-strand DNA breaks in lymphocytes [8]. Hexavalent chromium can also contribute to the formation of DNA-protein cross-links [5]. The primary sites of chromium absorption are lung and gastro-intestinal tract. The powder obtained from the plant Curcuma longa Linn, has been used for a long time as a naturally occurring medicine in the treatment of many diseases [9]. The main yellow pigment in this powder is curcumin (diferuloyl methane, Fig. 1) and it is also its major antioxidant and anti-inflammatory substance [10]. Curcumin is a commonly used food additive, especially in spices, mustard, and curry. Results from various animal tumor models demonstrate that curcumin can act as a chemopreventive agent. Administration of 0.5 to 2% curcumin in the diet of mice inhibited the incidence and size of colonic, small intestinal, and gastric cancers induced by azoxymethane [11,12]. The exact mechanism(s) underlying this effect is unknown. However, dietary curcumin can decrease the activity of phospholipase A2, cyclooxygenase, and lipoxygenase in both colonic mucosa and azoxymethane-induced tumors [11]. Curcumin can inhibit cell proliferation and induce cell cycle changes in colon adenocarcinoma cell lines by a prostaglandin-independent pathway [13]. It was also shown to inhibit the expression of oncogenes c-jun, c-fos, and cmyc in animals and cell lines [14,15] as well as the activity of the AP-1 transcription

DNA Damage After Exposure to Chromium and Curcumin

Fig. 1.

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Chemical structure of curcumin (diferuloyl methane).

factor in cells stimulated to proliferate [16]. It could protect DNA against strand breaks induced by singlet oxygen, a reactive oxygen species with potential genotoxic properties often created by the action of many chemicals [17]. Curcumin is reported to inhibit proliferation and apoptosis in human lymphoblastoid cells [18], which can have dual anti- and pro-carcinogenic meaning. On the other hand, curcumin was found to be cytotoxic in rat hepatocytes [19]. It was also shown to stimulate oxidative damage caused by quertecin in rat hepatocytes [20]. The gastrointestinal tract is the primary site of absorption of both chromium and curcumin, so it is reasonable to check the combined action of these agents in the human gastric mucosa (GM) cells. DNA damage induced by chemicals appears primarily in the form of alterations of the phosphate backbone, sugar, or base modifications such as alkylations, cross-links, or formation of bulky DNA adducts, which are substrates for DNA repair mechanisms. Transient DNA breaks arise in the second step as a consequence of repair and can be considered as important markers of genotoxicity [21]. The single cell gel electrophoresis (Comet assay) is a sensitive genotoxicity test to investigate DNA damage and repair [22–24]. In this technique a small number of cells suspended in a thin agarose gel on a microscope slide is lysed, electrophoresed, and stained with a fluorescent DNA-binding dye. The principle of the method is that fragmented DNA molecules can migrate more readily in an electric field than intact molecules. When cells are embedded in agarose and subsequently lysed to remove proteins, smaller DNA fragments are able to migrate away from the residual nucleus. After subsequent DNA staining with a fluorescent DNA-binding dye and visualisation by using a fluorescence microscope, the observed objects resemble comets with a head region containing undamaged DNA and a tail containing the broken DNA. The amount of DNA able to migrate and, to a lesser extent, the distance of migration are indications of the number of strand breaks present in that cell. Cells with an increased level of DNA damage display an increased migration of chromosomal DNA from the nucleus towards the anode. In the alkaline version of the comet assay, DNA single strand breaks and alkali labile sites become apparent, and the extent of DNA migration indicates the level of DNA breakage in the cell [22]. It has been shown that the Comet assay can detect a broad spectrum of mutagens [24]. In the present work the ability of curcumin to reduce DNA damage induced by chromium in the human GM cells, a potential target for both chromium and curcumin, was investigated using the alkaline comet assay. Because of exposure to chromium

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and curcumin can result in the presence of these agents in the blood, additional experiments with the human peripheral blood lymphocytes were performed. MATERIALS AND METHODS Cells Isolation

Human GM cells were obtained from macroscopically healthy tissue of donors as described elsewhere [25]. Briefly, biopsy samples were obtained during gastroscopy and were transported from the hospital to the laboratory in ice-cold Hanks’ balanced salt solution (HBSS) within 10 min. The samples were incubated in 1.5 ml digestion mixture comprised of 3 mg proteinase K and 2 mg collagenase in HBSS for 1 h at 37°C. The resulting mixture was centrifuged for 15 min at 160g and suspended in HBSS to give about 104 cells/ml. The viability of the cells was measured by the Trypan blue exclusion and it was constantly found to be about 88%. Blood was obtained from young, healthy, non-smoking donors. Peripheral blood lymphocytes were isolated by centrifugation in a density gradient of Gradisol L (15 min, 280g) [26]. The viability of the cells was measured as in the case of lymphocytes and it was found to be about 99%. Lymphocytes accounted for about 92% of leukocytes in obtained cell suspension. The final concentration of the lymphocytes was adjusted to 1–3 ´ 105 cells/ml by adding RPMI 1640 medium to the single cell suspension. Cells Treatment

Potassium dichromate, K2Cr2O7, was derived from a stock (20 mM) solution in phosphate-buffered saline (PBS) and added to the suspension of lymphocytes in RPMI 1640 medium or GM cells in HBSS to give a final concentration of 500 mM. Curcumin was taken from a stock (10 mM) ethanol solution and added to the suspension of either type of cells to give final concentrations of 15, 25, and 50 mM. The concentration of the chemicals in the working solutions was checked spectrophotometrically. The control cells were treated, instead of with chromium and curcumin, by PBS and ethanol in a concentration of 0.48%, respectively, not affecting the processes under study (data not shown). To examine DNA damage, the cells were incubated with either chemical or both chromium and curcumin for 1 h at 37°C. Each experiment included a positive control, which was hydrogen peroxide at 20 mM. H2O2 produced pronounced DNA damage, which resulted in comet tail moment of 80–120 mm (results not shown). Comet Assay

The Comet assay was performed under alkaline conditions essentially according to the procedure of Singh et al. [22] with a slight modification. A freshly prepared suspension of PBL in 0.75% low melting point agarose dissolved in PBS was casted onto fully frosted microscope slides (Superior, Germany) precoated with 0.5% normal melting agarose. The cells were then lysed for 1 h at 4°C in a buffer consisting of 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, and 10 mM Tris, pH 10. After the lysis, the slides were placed in an electrophoresis unit, allowing DNA to unwind for 40 min in the electrophoretic buffer, consisting of 300 mM NaOH, and 1 mM EDTA, pH > 13. Electrophoresis was conducted at an ambient temperature of 4°C (the temperature of the running buffer not exceeding 12°C) for 30 min at an electric field strength 0.73 V/cm (30 mA). The slides were then neutralized with 0.4 M Tris, pH 7.5, stained with 2 mg/ml DAPI,

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and covered with cover slips. To prevent additional damage all the steps described above were conducted under a dimmed light or in the dark. DNA Repair

To examine DNA repair, the cells after the treatment as well as control samples were washed and incubated in fresh, chromium- and curcumin-free RPMI 1640 medium or HBSS for 1 h at 37°C. Aliquots of the suspension were taken immediately and at 10, 30, and 60 min later. Placing the samples in an ice bath stopped the repair incubation. Comet Analysis

The objects were observed at ´200 magnification in a Labophot-2 fluorescence microscope (Nikon, Japan) attached to a Pulnix video camera (Kinetic Imaging, Liverpool, UK) equipped with a UV-1A filter block (an excitation filter of 365/10 nm and a barrier filter of 435 nm) and connected to a personal computer-based image analysis system Comet v. 3.0 (Kinetic Imaging, Liverpool, UK). Fifty images were randomly selected from each sample and the comet tail moment (a product of fraction of DNA in tail and tail length) was measured. Two parallel tests with aliquots of the same sample of cells were performed for a total of 50 cells and the mean comet length was calculated. The comet tail moment is positively correlated with the level of DNA breakage in a cell [22]. Because the distribution of the comets was heterogeneous, histograms were used to display information. The mean value of the tail moment in a particular sample was taken as an index of DNA damage in this sample. Statistics

All the values in this study were expressed as mean ± SEM from two separate experiments. The data were analyzed using STATISTICA (StatSoft, Tulsa, OK) statistical package. If no significant differences between variations were found, as assessed by the Snedecor-Fisher test, the differences between means were evaluated by applying Student t-test. Otherwise, the Cochran-Cox test was used. Chemicals

Potassium dichromate at purity of at least 99.5%, curcumin (diferuloyl methane), RPMI 1640 medium without glutamine, low melting point agarose, Hanks’ balanced salt solution (HBSS), phosphate-buffered saline (PBS), proteinase K, collagenase, and DAPI (4¢,6-diamidino-2-phenylindole) were obtained from Sigma Chemical Co. (St. Louis, MO). Gradisol L was from Polfa (Kutno, Poland). All other chemicals were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO). RESULTS DNA Damage

The mean comet tail moments for the lymphocytes and GM cells exposed for 1 h to chromium as compared with the appropriate controls are presented in Table I.

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TABLE I. Comet Tail Moment (mm) of Lymphocytes and Gastric Mucosa (GM) Cells Exposed to Chromium† Tail moment (mm) Treatment

Lymphocytes

GM cells

Control Potassium dichromate at 500 mM

9.82 ± 1.99 20.31 ± 1.91*

8.24 ± 2.09 23.40 ± 2.10*

The number of cells in each treatment was 50; mean ± SEM. *P < 0.001, relative to untreated control.



It can be seen from Table I that chromium at 500 mM evoked an increase in the comet tail moment of the lymphocytes of 105% as compared with the control (P < 0.001). The increase observed for the GM cells was 183% (P < 0.001). Table II shows the mean comet tail moments for the lymphocytes and GM cells exposed for 1 h to curcumin. Curcumin significantly increased the DNA migration at all tested concentrations, 15, 25, and 50 mM, in a dose-dependent manner: the comet tail moment of the exposed lymphocytes exceeding over two times (P < 0.01), over five times (P < 0.001), and nearly twenty times (P < 0.001), respectively, the comet tail moment of the control lymphocytes. Similar increase in the tail moment was observed in the case of GM cells. The combined action of chromium at 500 mM and curcumin at 50 mM resulted in the significant (P < 0.001) increase in the comet tail moment of both lymphocytes and GM cells (Table III). The effect of the two agents had roughly an additional character, as compared to the effects caused by either of them separately. The most basic way of viewing the data from the comet assay is the distribution of cells according to the percentage of DNA in the tail, which is positively correlated with the comet tail moment [27]. Figure 2 shows the distribution of GM cells according to their comet tail moments after treatment with chromium and curcumin. It can be seen from the figure that comets resulting from lymphocytes exposed to the agents used have greater tail moments, so they contain more DNA in their tails than comets resulting from control cells. The distribution of lymphocytes as well as GM in the remaining modes of exposition showed similar pattern. TABLE II. Comet Tail Moment (mm) of Lymphocytes and Gastric Mucosa (GM) Cells Exposed to Curcumin† Tail moment (mm) Treatment

Lymphocytes

Control Curcumin at 15 mM Curcumin at 25 mM Curcumin at 50 mM

2.05 ± 0.28 4.82 ± 0.99* 10.34 ± 1.89** 40.01 ± 3.22**

The number of cells in each treatment was 50; mean ± SEM. *P < 0.01. **P < 0.001, relative to untreated control.



GM cells 6.17 ± 1.52 14.31 ± 2.08* 30.26 ± 2.97** 51.23 ± 8.87**

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TABLE III. Comet Tail Moment (mm) of Lymphocytes and Gastric Mucosa (GM) Cells Exposed to Chromium and Curcumin† Tail moment (mm) Treatment

Lymphocytes

Control Potassium dichromate at 500 mM Curcumin at 50 mM Potassium dichromate at 500 mM and curcumin at 50 mM

4.21 ± 0.52 14.28 ± 1.56*** 43.48 ± 7.29*** 51.56 ± 4.80***

GM cells 7.80 ± 0.31 20.02 ± 4.50* 45.02 ± 10.01** 63.81 ± 11.22***

The number of cells in each treatment was 50; mean ± SEM. *P < 0.01. **P < 0.002. ***P < 0.001, relative to untreated control.



DNA Repair

Figure 3 shows the comet tail moments of lymphocytes exposed to chromium at 500 mM, curcumin at 50 mM, and both these agents immediately after the exposure as well as 10, 30, and 60 min thereafter. In all cases the comet tail moment of the control lymphocytes was constant, indicating that preparation and subsequent processing of the lymphocytes did not introduce a significant damage to their DNA. The lymphocytes exposed to 20 mM hydrogen peroxide (positive control) were able to recover within the repair incubation time of 60 min (results not shown). The lymphocytes exposed to chromium at 500 mM were able to remove the DNA damage within time period shorter than 30 min. The exposure to curcumin at 50 mM required the subsequent 60 min for almost full removal of DNA damage. The combined action of chromium and curcumin caused pronounced changes in the lymphocytes. The level of DNA damage after a 60 min post-treatment incubation, related to the comet tail moment, was significantly greater than those of the cells exposed to 500 mM chromium immediately after the exposure. However, evident tendency of the cell to recover can be deducted from the time course of the DNA repair displayed in Figure 3 and additional 10–20 min of repair incubation would allow the cell to remove completely DNA damage. DISCUSSION

Our experimental data indicate that single cell gel electrophoresis (comet assay) is a highly sensitive technique to analyse DNA damage induced by chromium and curcumin, which are reported to play a role in the carcinogenesis. The results obtained indicate that chromium and curcumin investigated with the comet assay give a significant increase in the tail moment of the comets of human peripheral blood lymphocytes and GM cells, acting therefore as genotoxic compounds. These results support the view of many authors, who have suggested the high sensitivity of this assay to detect DNA damage [22,28–30]. Besides DNA damage, strand-break formation during the process of excision repair may also cause DNA migration measurable in the comet assay [31,32]. Thus, DNA repair that reduces DNA damage by eliminating DNA lesions may, on the other hand, increase DNA migration due to incisionrelated strand breaks. The processes of repair can influence the results obtained with the comet assay in a complex way [33].

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Fig. 2. Histograms of the distribution of comet tail moments in human gastric mucosa cells treated with potassium dichromate at indicated concentrations as compared with control. The number of cells scored for each treatment was 50.

The present data indicate that hexavalent chromium and curcumin have an ability to damage DNA of isolated human peripheral blood lymphocytes and gastric mucosa cells. The concentrations of the chemicals used were not cytotoxic to the cells, as judged by Trypan blue exclusion and study of DNA repair kinetics. In the United Kingdom, occupational exposure limit for hexavalent chromium is 0.05 mg/ m3 as a maximum exposure limit [34], but the concentration of chromium in whole blood, plasma, and urine of chromate production workers can be much higher [35]. In chromate pigment production workers, urinary chromium was reported as 6–121 mg/g creatinine, 20–214 mg/l for blood, and 25–105 mg/l for serum [36, 37]. The concentration range of curcumin we used was typical for other research [17]. Although the main absorption sites of chromium in human are lungs, but this agent is also deposited in gastrointestinal tract so GM cells and subsequently blood can be considered as absorption sites for both chromium and curcumin. Chromium was proved to have genotoxic and/or carcinogenic properties in many studies [18–

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Fig.3. Time course of the repair of DNA damage in human lymphocytes and gastric mucosa cells treated with chromium at 500 mM (closed circle), curcumin at 50 mM (open square) and both chromium and curcumin (closed square) as compared with control (open circle). The number of cells scored for each treatment was 50. Error bars denote SEM.

20], including those with humans in vivo [8,38,39]. The results on chromium obtained in our study are in general agreement with those cited above and many others. However, our results that demonstrate that curcumin is able to damage DNA of the intact cell is rather surprising. A well-established view is that curcumin has antioxidant and anticarcinogenic properties [12]. In animal model systems, curcumin was shown to reduce the occurrence of benzo(a)pyrene [B(a)P]-induced forestomach

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tumor in mice [13] and inhibit 7,12-dimethylbenz(a)anthracene (DMBA)-induced hyperplastic nodules in cultured rat mammary gland tissues [40] and azoxymethaneinduced crypts in rat colon [41]. On the other hand, curcumin was also reported to stimulate of quercetin-induced DNA damage in isolated rat-liver nuclei [23] and exert cytotoxic effect in rat hepatocytes [22]. Curcumin can influence carcinogenesis by several mechanisms. It can inhibit the metabolic activation and promote the detoxification of carcinogens, as was shown for the inhibition of metabolic activation of B(a)P to mutagens and metabolic activation of B(a)P to B(a)P-DNA adducts in mouse skin in vitro [42], and the formation of B(a)P-DNA adducts or single strand breaks in DNA in the forestomach or liver of mice [43]. Curcumin was also shown to increase the rate of DNA repair in yeast [44]. The reduction of cancer cell number by curcumin can be done by the induction of apoptosis or by antiproliferative effect [12,45]. Apoptosis results in the extensive formation of double-strand breaks and is readily detected by the Comet assay by using either neutral or alkaline electrophoretic conditions [46]. Comets resulting from apoptosis have almost all DNA in their tails with very little or no DNA in their heads [32]. Comets typical for apoptosis were not observed in our study, so the reported DNA damage cannot be connected with apoptosis. It is also very unlikely that observed curcumin-induced DNA damages could play a positive role in carcinogenesis by causing potential reverse mutations in some genes often mutated in cancer (oncogenes, tumor suppressor genes, and mutator genes). The only reasonable explanation of our results is that curcumin may play a dual role in carcinogenesis and in some conditions, which need to be established, may be carcinogenic. Our study was not designed to search for the mechanism(s) underlying observed genotoxic effects of chromium and curcumin. In the case of chromium, the mechanism is rather well established and was outlined in the Introduction, but in light of data on anticarcinogenic action of curcumin, it is hard to speculate on possible mechanism that can contribute to the direct DNA-damaging action of curcumin. However, some aspects of interaction between curcumin and DNA can be considered. It is conceivable that the molecular structure of curcumin (Fig. 1) allows it to undergo autoxidation in the presence of oxygen and transition metal ions. This statement is supported by the results on stimulation of DNA damage by curcumin, which took place only in the presence of copper [23]. Thus, curcumin has the potential of acting as both pro- and anti-oxidant and in the complex cellular environment these two effects may be opposite. It is very important to establish circumstances of the particular oxidant mode of this agent. It seems first of all that the redox state of the biological environment of curcumin should be taken into consideration [23]. Our results clearly demonstrate that curcumin does not inhibit DNA damaging action of hexavalent chromium in human lymphocytes and GM cells. Moreover, curcumin itself can damage DNA of these cells and the total effect of chromium and curcumin is additive. Further studies are needed to establish the role of the interaction of curcumin with DNA in carcinogenesis. CONCLUSIONS

Single cell gel electrophoresis (the Comet assay) is a highly sensitive technique to analyse DNA damage and repair induced by agents, which are reported to play a role in the mutagenesis/carcinogenesis. Curcumin, reported to have anticarcinogenic

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properties in many studies, does not inhibit DNA damaging action of hexavalent chromium in human lymphocytes and gastric mucosa cells, which is a well-recognized carcinogen. Curcumin itself induces DNA damage in these cells, so it may have a dual role in carcinogenesis and this address warrants further investigation in order to determine the mode of action of curcumin in dependence on its biological environment. ACKNOWLEDGMENTS

This work was supported by grant 505/674 from University of Lodz (J.B.), and grant 502-11-422 (119) from Medical University of Lodz (E.M.-P.). Thanks to Ewa Jaruga. REFERENCES 1. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Chromium, nickel and welding. IARC 1990;49:447–525. 2. Cohen MD, Kargacin B, Klein CB, Costa M. Mechanisms of chromium carcinogenicity and toxicity. Crit Rev Toxicol 1993;23:255–281. 3. Susa N, Ueno S, Furukawa Y, Ueda J, Sugiyama M. Potent protective effect of melatonin on chromium(VI)-induced DNA single-strand breaks, cytotoxicity, and lipid peroxidation in primary cultures of rat hepatocytes. Toxicol Appl Pharmacol 1997;144:377–384. 4. Coogan TP, Mots J, Snyder CA, Squibb KS, Costa M. Differential DNA-protein crosslinking in lymphocytes and liver following chronic drinking water exposure of rat to potassium chromate. Toxicol Appl Pharmacol 1991;109:60–72. 5. De Flora S, Wetterhahn KE. Mechanism of chromium (VI) metabolism and genotoxicity. Life Chem Rep 1989;7:169–244. 6. Sugiyama M. Role of cellular antioxidants in metal-induced damage. Cell Biol Toxicol 1994;10:1–22. 7. Ueno S, Sugiyama M, Susa N, Furukawa Y. Effect of dimethylthiourea on chromium (VI)-induced DNA single-strand breaks in Chinese hamster V-79 cells. Mutat Res 1995;346:23–31. 8. Werfel U, Langen VI, Eickhoff I, Schoonbrood J, Vahrenholz C, Brauksiepe A, Popp W, Norpoth K. Elevated DNA single-strand breakage frequencies in lymphocytes of welders exposed to chromium and nickel. Carcinogenesis 1998;19:413–418. 9. Chopra RN, Chopra IC, Handa KI, Kapur LD. Indigenous Drugs of India. Calcutta: Dhur, 1958;325–327. 10. Sharma OP. Antioxidant of curcumin and related compounds. Biochem Pharmacol 1976;25:1811– 1812. 11. Rao CV, Rivenson A, Simi B, Reddy BS. Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound. Cancer Res 1995;55:259–266. 12. Hanif R, Qiao L, Sfiff SJ, Rigas B. Curcumin, a natural plant phenolic food additive, inhibits cell proliferation and induces cell cycle changes in colon adenocarcinoma cell lines by a prostaglandin-independent pathway. J Lab Clin Med 1997;130:576–584. 13. Nagabhushan M, Bhide SV. Curcumin as an inhibitor of cancer. J Am Coll Nutr 1992;11:192–198. 14. Lu YP, Chang RL, Lou Y, Huang MT, Newmark HL, Reuhl KR, Conney AH. Effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate and ultraviolet B light-induced expression of c-Jun and c-Fos in JB6 cells and in the mouse epiderms. Carcinogenesis 1994;15:2263–2370. 15. Kakar SS, Roy D. Curcumin inhibits TPA induced expression of c-fos, c-jun and c-myc protooncogene messenger RNAs in mouse skin. Cancer Lett 1994;87:85–89. 16. Huang TZ, Lee SC, Lin JK. Suppression of c-Jun/AP-1 activation by an inhibitor of tumor promotion in mouse fibroblast cells. Proc Natl Acad Sci USA 1991;88:5292–5296. 17. Subramanian M, Sreejayan M, Rao MNA, Devasagayam TPA, Singh BB. Diminution of singlet oxygen-induced DNA damage by curcumin and related antioxidants. Mutat Res 1994;311:249–255. 18. Bagchi D, Vuchetich PJ, Bagchi M, Hassoun EA, Tran MX, Tang L, Stohs SJ. Induction of oxidative stress by chronic administration of sodium dichromate (chromium VI) and cadmium chloride (cadmium II) to rats. Free Radic Biol Med 1997;22:471–478.

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