Mutagenesis, Vol. 16, No. 3, 265-270,
May 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Is the capacity of lead acetate and cadmium chloride to induce genotoxic damage due to direct DNA–metal interaction?
Departamento de Genética y Toxicología Ambiental, Instituto de Investigaciones Biomédicas and 1 Instituto de Fisiología Celular, UNAM Ciudad Universitaria, CP 04510, México D.F., Mexico
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Even though the toxic effects of lead and cadmium compounds have been studied over many years, inconsistent results have been obtained about their mutagenic, clastogenic and carcinogenic properties. However, these metals are considered to be potential human carcinogens. The mechanism of metal-induced carcinogenesis is still unknown, but one possible pathway may involve the interaction of metals with DNA, either directly or indirectly. In this work we explore the capacity of lead, cadmium or a mixture of both metals to interact with acellular DNA, by employing a variant of the comet assay. Our results, using low non-cytotoxic metal concentrations (0.01, 0.1 and 1.0 µM) with the standard protocol for the acellular assay, showed an induction of DNA damage in cells of all organs studied; however, basal DNA damage was different in each organ. To confirm that we were working with pure DNA, proteinase K was added to the lysis solution. With this enriched-lysis solution we found a negative response in the induction of DNA damage in cells derived from the liver, kidney and lung of CD-1 male mice. To support the results obtained by the enriched-acellular assay, we studied the capacity of lead and cadmium (0.1 µM) to induce breaks in pooled genomic DNA in cells of the same organs, with negative results. Consistent with these findings, these metals do not induce DNA breaks in the plasmid pUSE amp+. On the whole, we did not detect direct induction of DNA strand breaks by lead acetate, cadmium chloride or a mixture of both metals, all at low non-cytotoxic concentrations. However, we found an induction of lipid peroxidation and an increase in free radical levels in the different organs of CD-1 male mice after inhalation of lead acetate (0.0068 µg/cc) or cadmium chloride (0.08 µg/cc) for 1 h, suggesting the induction of genotoxicity and carcinogenicity by indirect interactions, such as oxidative stress.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
There is clear evidence that some metals represent a carcinogenic hazard to man. Cancer epidemiology has identified several metal compounds as human carcinogens (Frieberg et al., 1986; IARC, 1987
; IARC, 1993). However, the epidemiological evidence for carcinogenicity caused by lead compounds in humans is still inconsistent, and for cadmium compounds is complicated by confounders (Hartwig, 1994).
Regarding their genotoxicity in bacterial and mammalian cells, the results are conflicting, as neither lead (Pb) nor cadmium (Cd) are mutagenic in bacteria, whereas in mammalian cells they produce weak mutagenic effects (Rossman et al., 1992).
In this context, there are some studies that evaluated the direct and indirect genotoxic effects induced by lead and cadmium compounds in several tests. Lead is clearly not a powerful mutagen, nor is it consistently mutagenic among various test systems (Rossman and Molina, 1986; Zelikoff et al., 1988; Hartwig, 1994). Leonard (1988), after reviewing the literature, concluded that Pb compounds are devoid of clastogenic properties. On the other hand, Pb has been tested and found to be capable of eliciting a positive response in an extraordinarily wide range of biological and biochemical tests, including measures of DNA synthesis, mutation and chromosomal aberrations. However, Pb is considered to be a co-mutagen or a weak genotoxic agent (Rojas et al., 1999).
Cadmium scores negative in most bacterial short-term genotoxicity assays, but induces deletions in Saccharomyces cerevisiae (Brennan and Schiestl, 1996). Cadmium also induces DNA strand breaks, sister chromatid exchanges and chromosomal aberrations in plant, mammalian and human cells (Gebhart and Rossman, 1991; Forni, 1994; Hartwig, 1994; Misra et al., 1998; Saplakoglu and Iscan, 1998). This metal exerts pronounced indirect genotoxic effects: it enhances the mutagenicity of UV light in several cell types. Furthermore, at low non-cytotoxic concentrations, Cd inhibits unscheduled DNA synthesis after UV irradiation and partially inhibits the removal of UV-induced DNA lesions, suggesting an interference with DNA repair processes at relevant biological concentrations (Rojas et al., 1999).
Previous results from our group have shown an induction of genotoxicity in several organs of mice after chronic inhalation of both metals (Anderson et al., 1998; Valverde et al., 2000). Recent evidence suggests that the genotoxic effects could be the result of several mechanisms, such as the induction of cellular immunity and oxidative stress, the inhibition of DNA metabolism and repair, and the formation of DNA and/or protein cross-links (Snow, 1992).
Even though many metals are ubiquitous in our environment and some of these are designated as mutagens and carcinogens, little is known about their interactions with cellular macromolecules. Consequently, the mechanism of metal-induced carcinogenesis is still unknown, but one possible pathway may involve the interaction of metals with DNA, either directly or indirectly (Hartmann and Speit, 1994). The association of metal ions with DNA usually involves bonding by non-covalent interactions and these have been quantitated by a variety of physical techniques (Schaaper et al., 1987). Lead interacts with the phosphate backbone (Tajmir-Riahi et al., 1993), whereas cadmium preferentially forms interactions with nucleic acid bases over DNA phosphates (Jacobson and Turner, 1980; Koizumi and Waalkes, 1990). To discriminate the mechanism by which lead and cadmium are genotoxic, the aims of this work are to evaluate the capacity of these metals to interact with acellular DNA and to induce single strand breaks. For this purpose, we employed a version of the comet assay, based on the treatment of the liberated DNA present in the agarose gels after lysis (Kasamatsu et al., 1996; Tice et al., 2000). Considering that DNA and not cells are exposed, an alteration in DNA migration under these conditions indicates the ability of the test substances, in this case Pb and Cd, to induce DNA damage independent of cytotoxicity.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Chemical reagents
Normal agarose, low melting point agarose (LMPA), ethidium bromide, Tris, Na2EDTA, dimethyl sulfoxide (DMSO), Triton X-100, proteinase K and albumin were obtained from Sigma (St Louis, MO); NaOH, lead acetate and cadmium chloride from Merck (Germany); NaCl, KCl, Na2HPO4, KH2HPO4, absolute ethanol and acetic acid from Baker (Mex); loading buffer from BioRad (KA); Tripan blue, DNAzol, 1 kb DNA extension ladder from Gibco-BRL, NY and pUSE amp+ plasmid, containing the gene for I
B (7.3 kb), from Upstate Technology, NY.
Animals
Fifteen CD-1 male mice (from the Biomedical Research Institute Vivarium, UNAM, México), 45–63 days old and weighing 30–35 g, were housed in hanging plastic cages under controlled lighting conditions (12 h light/dark regime), relative humidity 50 ± 5% and temperature 17 ± 2°C and fed purine rat chow and water ad libitum.
Treatments
Inhalations were performed in an acrylic box (50x30x20.98 cm) connected to an ultra-nebulizer (Ultra-Neb 99; De Vilbis) with a flux of 10 l/min. For the exposure protocol, lead acetate and cadmium chloride concentrations were according to Fortoul et al. (1999). Briefly, nine animals, three from each group (Pb, Cd and Pb/Cd) were placed in an acrylic box and lead acetate (0.0068 µg/cc) and cadmium chloride (0.08 µg/cc) inhalations were performed for 60 min. The control animals inhaled deionized water at separate time. After inhalation, the animals were killed by cervical dislocation. The organs obtained (lung, liver and kidney) were homogenized in buffer containing 250 mM sucrose and 10 mM Tris–HCl (pH 7.4). Homogenates were used to determine lipid peroxidation and production of free radicals, procedures for which are described below.
Cell suspensions
Animals were killed by cervical dislocation, followed immediately by removal of liver, kidney and lungs into cold saline solution (0.9% NaCl). The tissues were then washed twice with cold saline solution and placed in cold PBS pH 7.4 and minced with the help of cold scissors into 1 mm3 pieces. Cell suspensions were then kept at 4°C until a sediment was observed.
The viability of the cell suspensions was determined using the trypan blue exclusion technique.
Acellular assay
An appropriate number of cells were obtained in 30 µl of cell suspension and mixed with 75 µl 0.5% LMPA. Cells and LMPA at 0.36% final concentration were loaded onto a microscope slide prelayered with 200 µl 0.5% normal agarose and allowed to gel at 4°C for 5 min. Then the slides were immersed in lysis solution at 4°C for 1 h [2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, fresh 1% Triton X-100 and 10% DMSO (pH 7)]. On these occasions when proteinase K (PK) was added, warm freshly made lysis solution (as before, pH 7) was prepared, when the lysis solution reached 37°C, 50 µl 20 mg/ml PK solution was added to a final concentration of 0.02 mg/ml. Slides were then incubated in this lysis solution for a further 1 h. In both cases, slides were washed twice in PBS pH 7.4 for 5 min each time in order to eliminate detergent.
For the treatment, the slides were immersed in 50 ml PBS pH 7.4 with or without test compounds (0.1 µM lead acetate, 0.1 µM cadmium chloride, or a mixture of both at the same concentration) for 10 min, a time sufficient for an interaction between the metals and acellular DNA. At the end of the treatment, slides were washed twice with PBS pH 7.4 and immediately placed in a horizontal electrophoresis chamber with running buffer (300 mM NaOH, 1 mM Na2EDTA, pH >13). The slides remained for 10 min in the electrophoresis buffer at 4°C to allow the DNA to unwind. Electrophoresis was performed for 10 min at 0.8 V/cm, and all technical steps were conducted using very dim indirect light. We previously determined that 10 min was the optimal electrophoresis time to obtain similar basal damage in all mouse organs employed (Valverde et al., 2000). After electrophoresis, the slides were gently removed and rinsed with neutralization buffer (0.4 M Tris–base, pH 7.5) at room temperature (15 min). The slides were dehydrated with absolute ethanol (15 min), after which they were air-dried. Ethidium bromide (75 µl of a 20 µg/ml solution) was added to each slide and a coverslip was placed on the gel. Individual cells were visualized at 200x magnification on an Olympus BX-60 microscope with fluorescence attachments (excitation filter 515–560 nm and barrier filter 590 nm) and the extent of migration (tail length value) was measured with a scaled ocular. For identifying tail image, the head of the comet was defined as the circular region more brilliant in the image. To evaluate DNA migration, 100 cells/tissue/animal were scored for each condition. Statistical analysis was performed using the Mann–Whitney U-test, which considered medians for analysis and the unit of the exposure and analysis used is the animal not the cells. Significance was at P < 0.05 (Kirk, 1999). All experiments were conducted in triplicate.
DNA isolation of tissues and DNA fragmentation analysis
During isolation, a small amount (5 mg) of soft tissue (lung, kidney and liver of CD-1 mice) was lysed or homogenized in DNAzol reagent and the genomic DNA was precipitated from the lysate with ethanol. Following an ethanol wash, DNA was solubilized in water. All experiments were performed in the dark. The standard reaction mixture contained 3 µg genomic DNA, 0.1 µM lead acetate or 0.1 µM cadmium chloride in TAE buffer (40 mM Tris, 1.14 ml/l acetic acid, 1 mM EDTA, pH 8, containing 2 µl 10 mg/ml ethidium bromide). The reaction mixture, final volume 20 µl, was incubated for up to 10 min at room temperature in polystyrene tubes. Aliquots of 18 µl were taken to analyse for DNA strand breaks; the aliquots were mixed with 2 µl loading buffer and loaded onto a 1% agarose TAE gel (pH 8). Electrophoresis was performed at 10 V/cm in TAE buffer, pH 8, for the neutral conditions, whereas alkaline conditions consisted of TAE buffer at pH 12 for the gel and electrophoresis buffer. The DNA was visualized on 312 UV-transiluminator and photographed using a Polaroid camera.
pDNA fragmentation
The assay for pDNA strand breaks was performed by exposing supercoiled plasmid DNA to a combination of lead acetate, cadmium chloride, or a mixture of both. The experiments were performed in the dark. The standard reaction mixture contained 1 µg/µl of the plasmid pUSE amp+, which contains the IB gene (7.3 kb), 0.1 µM lead acetate, or 0.1 µM cadmium chloride, or a mixture of both (0.1 µM each) in TAE buffer, pH 8.0. Reactions, with a final volume of 15 µl, were incubated for up to 10 and 30 min at room temperature in polystyrene tubes. Aliquots of 10 µl were taken to analyse the plasmid for the presence of DNA strand breaks. The aliquots were mixed with 2 µl loading buffer and loaded onto a 1.5% TAE agarose gel (pH 8, containing 2 µl 10 mg/ml ethidium bromide). Electrophoresis was performed at 10 V/cm in TAE buffer. The DNA was visualized on 312 UV-transilluminator and photographed using a Polaroid camera.
Lipid peroxidation and free radical measurement
We employed the method of Buege and Aust (1978). Malondialdehyde, formed from the breakdown of polyunsaturated fatty acids, serves as a convenient index for determining the extent of the peroxidation reaction. Malondialdehyde has been identified as the product of lipid peroxidation that reacts with thiobarbituric acid to give a red species absorbing at 532 nm.
Production of reactive oxygen species (ROS)
ROS induction was determined according to Cadenas and Sies (1984) using luminol (5-amino-2,3-dihydrophthalazine-1,4-dione). Luminol is employed to amplify chemiluminescence signals, this compound is oxidized by several oxygen intermediates to an electronically excited aminophthalate anion, that upon relaxation to the singlet ground state, emits photons. Emittance was measured using a liquid scintillation counter at 450 nm.
Statistical analysis
All statistical analysis was performed with GraphPAD inStat software ver. 1.14, USA. The Mann–Whitney U-test was used to determine statistical differences between treatments for each organ in the acellular assay. For production of free radicals and lipid peroxidation, ANOVA t-test was used to determine statistical differences between treatments for each organ; values of P < 0.05 were considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cell suspension viability, using the classical trypan blue exclusion technique, was greater than 80% in all organs immediately after killing.
DNA damage induced by lead acetate (0.01, 0.1 and 1 µM), cadmium chloride (0.01, 0.1 and 1 µM) or a mixture of both (0.1 µM lead acetate plus 0.1 µM cadmium chloride), using the acellular assay showed a heterogeneous response between lung, liver and kidney (Figure 1). We observed induction of DNA damage in cells of all organs but without an apparent dose–response. DNA damage induced by the lead acetate/cadmium chloride mixture showed a synergistic effect in lung and kidney, but not in liver (Figure 1).
|
When we employed the acellular assay, adding PK, we did not find induction of DNA single-strand breaks between treatments in the same organs (Figure 2
). Likewise, we observed a lack of synergistic effect under this condition.
|
In light of the conflicting results, we decided to treat genomic DNA, obtained from cells of each organ (Figure 3
), with the same metal concentration (0.1 µM) for 10 min. We did not detect an increase on DNA fragmentation either with Pb or Cd using either neutral or alkaline conditions (Figure 4).
|
|
To corroborate this finding, a smaller fragment of DNA was employed corresponding to the plasmid pUSE amp+, which contains the I
B gene (7.3 kb). Using this plasmid as a target of metal treatments, we did not find any induction of pDNA fragmentation, even if the time of treatment was increased up to 30 min (Figures 5 and 6).
|
B) (7.3 kb) non-treated (control), lane +Pb corresponds to the plasmid treated with 0.1 µM lead acetate, lane +Cd corresponds to plasmid treated with 0.1 µM cadmium chloride, and lane +Pb/Cd is the same plasmid treated with both 0.1 µM cadmium chloride plus 0.1 µM lead acetate.
|
With respect to the evaluation of indirect effects of metals, we determined the production of free radicals by luminol-dependent chemiluminescence. These data (Table I
) showed that inhalation of lead acetate (0.0068 µg/cc) produced higher levels of free radicals than cadmium chloride inhalation (0.08 µg/cc) in different organs of CD-1 male mice. The greatest effect was in liver, whereas kidney showed a negative response.
|
Likewise, we found that inhalation of lead acetate (0.0068 µg/cc) induced higher lipid peroxidation levels than cadmium chloride inhalation (0.08 µg/cc) only in cells of the lung; however, in liver cells we detected the same induction of lipid peroxidation levels for both metals (Table II
). In contrast, data in Table I
show levels of lipid peroxidation were higher in the lung, whereas in kidney induction was smaller (Table II).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Even though the toxic effects of lead and cadmium compounds have been studied over many years, inconsistent results have been published about their mutagenic, clastogenic and carcinogenic properties. Leonard and Bernard (1993) proposed that the response obtained was dependent on the ability of the metal to penetrate the cell and to interact with DNA. Others suggest that the mechanism of metal-induced carcinogenesis is still unknown, but one possible pathway may involve the interaction of metals with DNA, either directly or indirectly (Hartmann and Speit, 1994; Hwua and Yang, 1998
). The aim of this work was to explore the ability of lead and cadmium to interact directly with DNA, and to induce DNA single-strand breaks as a possible mechanism for the induction of genotoxicity observed previously (Valverde et al., 2000). For this purpose, we employed the acellular assay previously described by Kasamatsu (1996) to detect the induction of DNA damage obtained after lysing cells from several organs of the mouse.
Under these conditions, we observed a heterogeneous response to different concentrations of metals (0.01, 0.1, 1 µM) between DNA from cells of the lung, liver and kidney (Figure 1). These results were surprising because we expected the same level of DNA damage among different organs using acellular DNA. These results suggest the possible presence of proteins associated with DNA that could be modulating the metal genotoxic response, as reported by others (Scicchitano and Pegg, 1987; Tajmir-Riahi et al., 1993; Ovelönne et al., 1995; Dally and Hartwig, 1997). Thus, we modified the lysis conditions by preparing a new lysis solution containing proteinase K under optimal conditions (pH 7, 37°C for 1 h). Proteinase K has been reported as a powerful protease (Merk and Speit, 1998; Singh et al., 1989; Tice et al., 2000).
Using this protocol, we decided to test lead acetate and cadmium chloride at concentrations of 0.01, 0.1 and 1.0 µM, because in this range has been included the dose proposed as the permissible exposure limit (OSHA, 1981). These experiments did not show an induction of DNA damage by either metal in acellular DNA from cells of the lung, liver and kidney of CD-1 mice. This supports our assumption that the lysis solution is unable to obtain pure DNA, because when we added PK in the lysis solution, it was possible to observe a homogeneous response. In spite of this, we did not find induction of DNA damage. Our results show that lead acetate, cadmium chloride or a mixture of the two did not have the capacity to interact directly with DNA and produce single strand breaks.
These results are supported by our assays of DNA fragmentation of genomic DNA from cells of the lung, liver and kidney, performed in both neutral and alkaline conditions, because in both cases we did not detect fragmentation induced by Pb and Cd at low non-cytotoxic concentrations (0.1 µM). Likewise, pDNA fragmentation assays showed a negative response by the same treatments and the mixture.
These results disagree with the report of Yang et al. (1999), who found an induction of DNA strand breaks by lead acetate using a plasmid relaxation assay. The differences between this study and our own results could be explained by the concentration employed. Yang and co-workers used 10 mM lead acetate whereas our tested concentration was 0.1 µM [our concentration is equimolar equivalent to the permissible exposure limit approved by the OSHA (1981)]. This is in agreement with Hartwig (1998), who proposed that direct genotoxic effects induced by metals are rather weak and/or restricted to high concentrations.
Our results suggest that these metals did not interact directly with DNA. These results led us to two different possible explanations of the genotoxicity observed by inhalation of these metals: (i) the possibility of DNA-associated protein–metal interactions, or (ii) the induction of an indirect mechanism, such as oxidative stress.
To help resolve these possibilities we explored the induction of ROS and lipid peroxidation levels after inhalation of these metals (Tables I and II). We observed an induction of this kind of indirect effect after a single inhalation of lead acetate and cadmium chloride only in cells of the liver and lung. The absence of a response in kidney cells could be explained by a possible induction of pro-inflamatory cytokines, such as TNF
or interleukin 1 (IL-1), modulating the induction of oxidative stress (Oberdöster et al., 1994). However, another explanation is that a single inhalation of these metals did not have the capacity to induce an oxidative response. Our results are in agreement with Snyder (1988), Roy and Rossman (1992), Snow (1992), Kawanishi et al. (1994), Sarkar et al. (1998) and Yang et al. (1999), suggesting the implication of an oxidative response. In addition, other indirect mechanisms could be considered as Dally and Hartwig (1997) reported that cadmium at a concentration of 10 µM induced DNA strand breaks but without oxidative DNA base modification. Further studies are needed to define the role and pathway of this ROS to exert the genotoxic effects observed by the inhalation of these metals at low concentrations.
|
Acknowledgments |
|---|
This study was partially supported by CONACyT project 3180P-M. M.V. was the recipient of a fellowship from CONACyT and DGEP.
|
Notes |
|---|
2 To whom correspondence should be addressed. Email: emilior{at}servidor.unam.mx
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Anderson,D. and Plewa,M.J. (1998) The International Comet Assay Workshop. Mutagenesis, 13, 67–73.
Brennan,R.J. and Schiestl,R.H. (1996) Cadmium is an inducer of oxidative stress in yeast. Mutat. Res., 356, 171–178.[Web of Science][Medline]
Buege,J.A. and Aust,S.D. (1978) Microsomal lipid peroxidation. Methods Enzymol., 52, 302–310.[Medline]
Cadenas,E. and Sies,H. (1984) Low-level chemiluminescence as an indicator of singlet molecular oxygen in biological systems. Methods Enzymol., 105, 221–231.[Web of Science][Medline]
Dally,H. and Hartwig,A. (1997) Induction and repair inhibition of oxidative DNA damage by nickel (ii) and cadmium (II) in mammalian cells. Carcinogenesis, 18, 1021–1026.
Forni,A. (1994) Comparison of chromosome aberrations and micronuclei in testing genotoxicity in humans. Toxicol. Lett., 72, 185–190.[Web of Science][Medline]
Fortoul,T.I., Salgado,R.C., Moncada,S.G., Sanchez,I.G., Lopez,I.E., Espejel,G., Calderon,N.L. and Saldivar,L. (1999) Ultrastructural findings in the nonciliated bronchiolar cells (NCBC) after subacute inhalation of lead acetate. Acta Vet. Brno, 68, 51–55.
Friberg,L., Kjellström,T. and Nordberg,G.F. (1986) Cadmium. In Friberg,L., Nordberg,G.F and Vouk,V. (eds) Handbook on the Toxicology of Metals, 2nd Edn. Elsevier Science, Amsterdam, pp. 130–184.
Gebhart,E. and Rossman,T.G. (1991) Mutagenicity, Carcinogenicity, Teratogenicity. VCH Verlagsgesellschaft, Weinheim, pp. 617–640.
Hartman,A. and Speit,G. (1994) Comparative investigations of the genotoxic effects of metals in the single cell gel (SCG) assay and the sister chromatid exchange (SCE) test. Environ. Mol. Mutagen., 23, 299–305.[Web of Science][Medline]
Hartwig,A. (1994) Role of DNA repair inhibition in leadand cadmium-induced genotoxicity: a review. Environ. Health Perspect., 102 (suppl. 3), 45–50.
Hartwig,A. (1998) Carcinogenicity of metal compounds: possible role of DNA repair inhibition. Toxicol. Lett., 102–103, 235–239.
Hwua,Y.-S. and Yang,J.-L. (1998) Effect of 3-aminotriazole on anchorage independence and mutagenicity in cadmiumand lead-treated diploid human fibroblasts. Carcinogenesis, 19, 881–888.
IARC (1987) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Supplement 7. IARC, Lyon.
IARC (1993) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Beryllium, Cadmium, Mercury and Exposures in the Glass Manufacturing Industry. IARC Scientific Publications No. 58, IARC, Lyon.
Jacobson,K.B. and Turner,J.E. (1980) The interaction of cadmium and certain other metal ions with proteins and nucleic acids. Toxicology, 16, 1–37.[Web of Science][Medline]
Kasamatsu,T., Kohda,K. and Kawazoe,Y. (1996) Comparison of chemically induced DNA breakage in cellular and subcellular systems using the comet assay. Mutat. Res., 369, 1–6.[Web of Science][Medline]
Kawanishi,S., Inoue,S. and Yamamoto,K. (1994) Active oxygen species in DNA damage induced by carcinogenic metal compounds. Environ. Health Perspect., 102 (suppl. 3), 17–20.
Kirk,R.E. (1999) Statistics. An Introduction, 4th Edn. Harcourt Brace College Publishers, Orlando, FL.
Koizumi,T. and Waalkes,M.P. (1990) Effects of zinc on the binding of cadmium to DNA: assessment with testicular interstitial cell and calf thymus DNAs. Toxicol. In Vitro, 4, 51–55.
Leonard,A. (1988) Mechanisms in metal genotoxicity: the significance of in vitro approaches. Mutat. Res., 198, 321–326.[Web of Science][Medline]
Leonard,A. and Bernard,A. (1993) Biomonitoring exposure to metal compounds with carcinogenic properties. Environ. Health Perpct., 101 (suppl. 3), 127–133.
Merk,O. and Speit,G. (1998) Significance of formaldehyde-induced DNA–protein crosslinks for mutagenesis. Environ. Mol. Mutagen., 32, 260–268.[Web of Science][Medline]
Misra,R.R., Smith,G.T. and Waalkes,M.P. (1998) Evolution of the direct genotoxic potential of cadmium in four different rodent cell lines. Toxicology, 126, 103–114.[Web of Science][Medline]
OSHA (1981) OSHA Occupational Safety and Health Standards. Subpart 2, Code of Federal Regulations 29 (Part 1910.1000), Washington, DC, pp. 673–679.
Oberdöster,G., Cherian,M.G. and Baggs,R.B. (1994) Correlation between cadmium-induced pulmonary carcinogenicity, metallothionein expression and inflammatory processes: a species comparison. Environ. Health Perpect., 102 (suppl. 3), 257–263.
Ovelgönne,J.H., Souren,J.E.M., Wiegant,F.A.C. and Van Wijk,R. (1995) Relationship between cadmium-induced expression of heat shock genes, inhibition of protein synthesis and cell death. Toxicology, 99, 19–30.[Web of Science][Medline]
Rojas,E., Herrera,L.A., Poirier,L.A. and Ostrosky-Wegman,P. (1999) Are metals dietary carcinogens? Mutat. Res., 443, 157–181.[Web of Science][Medline]
Rossman,T.G. and Molina,M. (1986) The genetic toxicology of metal compounds: II. Enhancement of ultraviolet light-induced mutagenesis in E. coli WP2. Environ. Mutagen., 8, 263–271.[Web of Science][Medline]
Rossman,T.G., Roy,N.K. and Lin,W. (1992) Is cadmium genotoxic?. In Nordberg,G.F., Alessio,L. and Herber,R.F.M. (eds) Cadmium in the Human Environment: Toxicity and Carcinogenicity. IARC, Lyon.
Roy,N.K. and Rossman,T.G. (1992) Mutagenesis and comutagenesis by lead compounds. Mutat. Res., 298, 97–103.[Web of Science][Medline]
Sarkar,S., Yadav,P. and Bhatnagar,D. (1998) Lipid peroxidative damage on cadmium exposure and alterations in antioxidant system in rat erythrocytes: A study with relation to time. BioMetals, 11, 153–157.[Web of Science][Medline]
Scicchitano,D.A. and Pegg,A.E. (1987) Inhibition of O6-alkylguanine-DNA-alkyltransferase by metals. Muat. Res., 192, 207–210.
Singh,N.P., Danner,D.B., Tice,R.R., McCoy,M.T., Collins,G.D. and Schneider,E.L. (1989) Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp. Cell Res , 184, 461–470.[Web of Science][Medline]
Snow,E.T. (1992) Metal carcinogenesis: mechanistic implications. Pharmacol. Ther , 53, 31–65.[Web of Science][Medline]
Snyder,R.D. (1988) Role of active oxygen species in metal-induced DNA strand breakage in human diploid fibroblasts. Mutat. Res , 193, 237–246.[Web of Science][Medline]
Splakoglu,U. and Iscan,M. (1998) Sister chromatid exchanges in human lymphocytes treated in vitro with cadmium in G and S phase of their cell cycles. Mutat. Res , 412, 109–114.[Web of Science][Medline]
Tajmir-Riahi,H.A., Naoui,M. and Ahmad,R. (1993) The effects of Cu2+ and Pb2+ on the solution structure of calf thymus DNA: DNA condensation and denaturation studied by Fourier Transform IR difference spectroscopy. Biopolymers, 33, 1819–1827.[Web of Science][Medline]
Tice,R.R., Agurell,E., Anderson,D., Burlinson,B., Hartmann,A., Kobayashi,H., Miyamae,Y., Rojas,E., Ryu,J.-C. and Sasaku,Y.F. (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen., 35, 206–221.[Web of Science][Medline]
Valverde,M., Fortoul,T.I., Díaz-Barriga,F., Mejia,J. and Rojas,E. (2000) Induction of genotoxicity by cadmium chloride inhalation in several organs of CD-1 mice. Mutagenesis, 15, 109–114.
Yang,J.-L., Wang,L.-Ch., Chang,Ch.-Y. and Liu,T.-Y. (1999) Singlet oxygen is the major species participating in the induction of DNA strand breakage and 8-hydroxydeoxyguanosine adduct by lead acetate. Environ. Mol. Mutagen , 33, 194–201.[Web of Science][Medline]
Zelikoff,J.T., Li,J.H., Hartwig,A., Wang,W.W., Costa,M. and Rossman,T.G. (1988) Genotoxic toxicology of lead compounds. Carcinogenesis, 9, 1727–1732.
Received on July 28, 2000; revised on December 22, 2000; accepted on April 1, 2001.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Alghazal, I Sutiakova, N Kovalkovicova, J Legath, M Falis, J Pistl, R Sabo, K Benova, L Sabova, and P Vaczi Induction of micronuclei in rat bone marrow after chronic exposure to lead acetate trihydrate Toxicology and Industrial Health, October 1, 2008; 24(9): 587 - 593. [Abstract] [PDF]
|
|
|
|
|
|
A. Celik, U. Comelekoglu, and S. Yalin A study on the investigation of cadmium chloride genotoxicity in rat bone marrow using micronucleus test and chromosome aberration analysis Toxicology and Industrial Health, October 1, 2005; 21(9): 243 - 248. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||