Effect of soluble and particulate nickel compounds on the formation and repair of stable benzo[a]pyrene DNA adducts in human lung cells

Tanja Schwerdtle1, Albrecht Seidel2 and Andrea Hartwig1,3

1 Institut für Lebensmittelchemie und Toxikologie, Universität Karlsruhe, D-76128 Karlsruhe and
2 Biochemisches Institut für Umweltcarcinogene, Prof Dr G.Grimmer-Stiftung, D-22927 Grosshansdorf, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nickel compounds are well-known human carcinogens, but the underlying mechanisms are still not fully understood. Even though only weakly mutagenic, nickel chloride has been shown previously to impair the repair of UV-induced DNA damage as well as oxidative DNA damage. However, the carcinogenic potential depends largely on solubility, with poorly water-soluble nickel subsulfide and nickel oxide being strong carcinogens. Within the present study we investigated the effects of particulate black NiO and soluble NiCl2 on the induction and removal of stable DNA adducts formed by benzo[a]pyrene (B[a]P) measured by a highly sensitive high performance liquid chromatography (HPLC)/fluorescence assay. With respect to adduct formation, NiO but not NiCl2 reduced the generation of DNA lesions by ~30%. Regarding their repair, in the absence of nickel compounds, most lesions were removed within 24 h; nevertheless, between 20 and 35% of induced adducts remained even 48 h after treatment. NiCl2 and NiO reduced the removal of adducts in a dose-dependent manner. Thus, 100 µM NiCl2 led to ~80% residual repair capacity; after 500 µM the repair was reduced to ~36%. Also, even at the completely non-cytotoxic concentration of 0.5 µg/cm2 black NiO, lesion removal was reduced to ~35% of control and to 15% at 2.0 µg/cm2. Furthermore, both nickel compounds increased the benzo[a]pyrene-7,8-diol 9,10-epoxide (BPDE)-induced cytotoxicity. Taken together, our results indicate that the nucleotide excision repair pathway is affected in general by water-soluble and particulate nickel compounds and provide further evidence that DNA repair inhibition may be one predominant mechanism in nickel-induced carcinogenicity.

Abbreviations: B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene-7,8-diol 9,10-epoxide; DMEM, Dulbecco's modified eagle medium; GG-NER, global genome nucleotide excision repair; HPLC, high performance liquid chromatography; MEM, minimal essential medium; NER, nucleotide excision repair; PAH, polycyclic aromatic hydrocarbons; TC-NER, transcription-coupled nucleotide excision repair; tetrol I-1, r-7,t-8,t-9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; THF, tetrahydrofuran


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nickel compounds have long been recognized as human and/or animal carcinogens (1,2). However, the underlying molecular mechanisms of nickel carcinogenicity are not well understood, especially because of the missing mutagenicity in bacterial test systems and only weak mutagenic effects in mammalian cells in culture (1). Very strikingly, the carcinogenic potential depends largely on solubility: While particulate nickel compounds with intermediate water solubility such as Ni3S2 are strong carcinogens, soluble nickel(II) salts exert weaker effects (1,3). This may be due to differences in bioavailability. Water-soluble nickel salts are taken up only slowly by cells, while particulate nickel compounds are phagocytosed and, due to the low pH, are gradually dissolved in lysosomes, yielding high concentrations of nickel ions in the nucleus (4). It cannot be excluded, however, that diverse mechanisms account for the carcinogenic potencies of the respective nickel compounds. Several approaches during recent years identified levels of interaction which may be relevant for the carcinogenic process. They include the induction of oxidative DNA damage (5,6), changes in DNA methylation patterns (7) and the interference with different DNA repair systems. With respect to the latter, nickel chloride has been shown to increase the UV-induced mutation frequencies in V79 Chinese hamster cells (8). Subsequent investigations revealed an interference with the repair of UVC-induced DNA lesions in HeLa cells (9), presumably due to a diminished DNA damage recognition during nucleotide excision repair (NER) (10). Furthermore, nickel chloride inhibited the repair of oxidative DNA base modifications and DNA strand breaks in the same cell line (11). These inhibitions have been observed at low concentrations, where the viability of the cells was not affected. The present study was undertaken to elucidate whether poorly water-soluble black NiO also exerts repair inhibition at non-cytotoxic concentrations. This compound has been shown to be phagocytosed by cultured cells, and measurable amounts of nickel have been detected in the cytoplasm and nuclei (12). In animal carcinogenicity studies, it caused a dose-related increase in alveolar and broncheolar neoplasms in rats (3). As a model repair system, we investigated the removal of stable DNA adducts formed by the carcinogen benzo[a]pyrene (B[a]P). B[a]P belongs to the class of polycyclic aromatic hydrocarbons (PAH) continuously formed during incomplete combustion of organic matter. Studying the effects of nickel compounds on DNA adducts of PAH such as those of B[a]P is of interest, because humans are frequently exposed to both classes of chemicals under occupational and environmental conditions (13).

The carcinogenic activity of B[a]P is attributed to the formation of DNA adducts, resulting from electrophilic attack predominantly at guanine residues by metabolically activated intermediates formed from the parent hydrocarbon. Routes of metabolic activation include the formation of radical cations via P450 and/or peroxidases and the formation of o-quinones via dihydrodiol dehydrogenases. For carcinogenicity, probably the most relevant metabolic pathway is connected to the action of cytochromes P450 1A1 and 1B1 and epoxide hydrolase, yielding syn- and anti-B[a]P-7,8-diol 9,10-epoxides (BPDE), which form adducts at the N2 position of guanine. In contrast to B[a]P-induced lesions at positions N7 or N8 of purines derived from radical cations, which give rise to apurinic sites due to hydrolysis of the N-glycosidic bond, hydrolysis of BPDE-induced DNA adducts is very slow and they are considered to be stable on cellular conditions. When replicated prior to repair, these adducts can lead to mutations and cancer (14–17). Thus, within the present study, the induction and removal of the latter adducts in the absence and presence of nickel compounds was quantified; to avoid an interference by nickel with the metabolism of B[a]P, the active metabolites (±)-anti-BPDE and (+)-anti-BPDE were applied. The experiments were performed in human HeLa cells frequently used for repair studies and in A549 human lung cancer cells, which retained important characteristics of pneumocytes type II with respect to metabolism and phagocytotic capability (18–20).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Dulbecco's modified eagle medium (DMEM), minimal essential medium (MEM), fetal calf serum and penicillin–streptomycin solutions are products of Gibco (Karlsruhe, Germany). Trypsin was obtained from Sigma (Munich, Germany). Calf thymus DNA and RNase A were purchased from Boehringer (Mannheim, Germany). Proteinase K and Giemsa stain were bought from Merck (Darmstadt, Germany), phenol/chloroform/isoamyl alcohol from Roth (Karlsruhe, Germany). HPLC-grade water and methanol were from Riedel-de Haën (Seelze, Germany).

All other chemicals were of p.a. grade and were obtained from Fluka Chemie (Buchs, Germany). The culture dishes were supplied by Biochrom (Berlin, Germany). r-7,t-8,t-9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo [a]pyrene (tetrol I-1) was obtained from the NCI Chemical Carcinogen Reference Standard Repository (Kansas City, USA). Black NiO was a kind gift of Dr A.Oller, Nickel Producers Environmental Research Association (Durham, USA). Particle size was determined by scanning electron microscopy and found to be <5 µm, with most particles being in the range of 0.5 to 3 µm.

Synthesis of (±)- and (+)-anti-benzo[a]pyrene 7,8-diol 9,10-epoxide
(±)-anti-BPDE was synthesized from racemic benzo[a]pyrene-7,8-dihydrodiol with m-chloroperbenzoic acid according to a published procedure (21). (+)-anti-BPDE was synthesized from (-)-benzo[a]pyrene-7,8-dihydrodiol as described for the racemic compound (21). (-)-Benzo[a]pyrene-7,8-dihydrodiol was obtained by chromatographic separation of diastereomeric esters as described below. Racemic benzo[a]pyrene-7,8-dihydrodiol was synthesized from pyrene as previously reported (22, 23), and for resolution of enantiomers it was converted into the diastereomeric bis-(-)-menthoxyacetate derivatives (24, 25). Separation of the diastereomeric mixture was performed by preparative HPLC using a Labomatic HD 200 chromatograph equipped with a 32 x 250 mm silica gel column (LiChrosorb Si 60, 5 µm, Knauer). Elution with 5% diethyl ether in cyclohexane, a solvent system earlier used by Yagi et al. (26) for similar separations, allowed the resolution of 50 mg samples of the bis-(-)-menthoxyacetates injected in the same solvent. The pure diastereomeric esters obtained after one rechromatography were saponified in methanol/tetrahydrofuran (THF) with sodium methoxide to give the (+)- and (-)-enantiomer of the benzo[a]pyrene-7,8-dihydrodiol. The specific rotation values of optically active benzo[a]pyrene-7,8-dihydrodiol and (+)-anti-BPDE were in good accordance to those reported by Yagi et al. (27).

Cell culture and incubation
A549 cells were grown as monolayers in Dulbecco's modified eagle medium (DMEM) containing 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. HeLa cells were grown as monolayers in minimal essential medium (MEM) containing 10% fetal bovine serum, 100U/ml penicillin and 100 µg/ml streptomycin. The cultures were incubated at 37°C with 5% CO2 in air and 100% humidification.

(±)-anti-BPDE and (+)-anti-BPDE were dissolved in water-free THF/5% triethylamine (1 µg/ml) and stored at -80°C. To avoid hydrolysis of the epoxides, dilutions from the stock solution were always prepared with fresh solvent (water-free THF/1% triethylamine) immediately before incubation. The final concentration of the solvent in medium was always 0.1%.

Directly before each experiment, particles of black NiO were sterilized for 30 min at 110°C and suspended by sonification for 20 min in bidistilled water. Soluble NiCl2 was dissolved in bidistilled water.

Colony-forming ability
Logarithmically growing cells were treated as described for the respective experiments, trypsinized and 300 cells/dish were seeded. After 7 days of incubation, colonies were fixed with ethanol, stained with Giemsa (25% in ethanol), counted and calculated as percent of control. Untreated controls exhibited colony-forming abilities of ~80%.

DNA isolation and purification
2–4 x 106 logarithmically growing cells were trypsinized, washed twice with ice-cold Tris-buffered saline (0.0027 M KCl, 0.137 M NaCl, 0.025 M Tris–base, pH 7.4) and collected by centrifugation. DNA was isolated as described previously (28) with modifications. After 1 h incubation with extraction buffer (10 mM Tris–HCl, 0.1 M EDTA, 0.5% SDS, 20 µg/ml DNase-free RNase A, pH 8.0) at 37°C, proteinase K was added to a final concentration of 100 µg/ml and the suspension of lysed cells was incubated at 50°C for 3 h. Thereafter, DNA was extracted at least twice with phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform/isoamyl alcohol (24:1). DNA was precipitated with ethanol/ammonium acetate and washed at least four times with 70% ethanol. DNA concentration was determined spectrophotometrically by UV absorbance at 260 nm. Absorbance ratios at 260/230 and 260/280, reflecting the purity of DNA, were always ~2.3 and >1.85, respectively.

HPLC/fluorescence analysis of tetrol I-1
We applied a procedure described previously (29,30) with modifications. Briefly, adducted DNA (10–100 µg) was hydrolysed by incubation at 90°C for 4 h in a final concentration of 0.1 N HCl and neutralized with NaOH to pH 7.0–7.5. To avoid the presence of interfering fluorescent compounds, HCl and NaOH were checked by HPLC and sample vials were washed repeatedly with HPLC-grade water. HPLC analysis was performed with a Gynkotek Gina 50 autosampler coupled to a Gynkotek M480 gradient pump. Tetrol I-1 was quantified by a Gynkotek RF 2000 fluorescence detector at an excitation wavelength of 344 nm and an emission wavelength of 398 nm. First, a pre-column (Luna C18, 5 µm, 4.6 x 30 mm, Phenomenex) was equilibrated with water (10 min, flow-rate 1.0 ml/min) and loaded with 1 ml sample solution. After washing with 20% methanol in water (10 min, flow-rate 0.5 ml/min), the concentrated tetrol I-1 was eluted isocratically with 55% methanol in water (flow-rate 1.0 ml/min) and separated by applying a Gynkotek MSV 6 switching valve to a 4.6 x 250 mm C18 analytical column (Luna C18, 5 µM, Phenomenex). Quantification of tetrol I-1 in the sample was carried out by comparing the peak areas of samples with an external calibration curve. For calibration, 100 µg calf thymus DNA were spiked with 4–100 pg tetrol I-1 and hydrolysed as described above. When investigating repair over time periods of 24 h and 48 h, adduct levels have been corrected for cell division.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Cytotoxicity of BPDE
The cytotoxicity of (±)-anti-BPDE was determined by colony-forming ability after 2 h incubation (Figure 1Go). HeLa as well as A549 cells showed a concentration-dependent decrease of colony-forming ability, with HeLa cells being more sensitive. While in A549 cells 200 nM (±)-anti-BPDE reduced the colony-forming ability to ~50 %, it was diminished to nearly 10% in HeLa cells.



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Fig. 1. Cytotoxicity of (±)-anti-BPDE in A549 and HeLa cells. Logarithmically growing cells were treated with (±)-anti-BPDE for 2 h, trypsinized and reseeded for colony-forming ability. Control values (100%) refer to medium containing 0.1% solvent. The data represent mean values of 12 determinations ± SD. If error bars are not visible, they are smaller than the size of symbols.

 
Formation and repair of BPDE-induced DNA adducts
BPDE–DNA adduct levels were measured by a highly sensitive HPLC/fluorescence assay. The principle of the procedure consists in the hydrolysis of stable DNA adducts formed at the N2 position of guanine by 0.1 N HCl, yielding the corresponding tetrol I-1 (29,30). In a first step, the assay including sample preparation was optimized, calibrated and statistically evaluated on cell-free conditions. When adding different amounts of tetrol I-1 to calf thymus DNA, hydrolysing and neutralizing as described in Materials and methods, the minimum correlation coefficient of the calibration curve was 0.9991 and the mean coefficient of variation for independent analyses on different days was 3.62% (Figure 2Go). Representative HPLC profiles of tetrol I-1 formed after acid hydrolysis of calf thymus DNA spiked with tetrol I-1 (A) or HeLa cells incubated with 50 nM (±)-anti-BPDE for 2 h (B) are shown in Figure 3Go. Under our conditions, the assay exerts a detection limit of 1 pg of tetrol I-1, requires 10–100 µg DNA and detects 1 adduct/108 base pairs after incubation of cells.



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Fig. 2. Calibration curve of tetrol I-1. 100 µg calf thymus DNA were spiked with 4–100 pg tetrol I-1, hydrolysed, neutralized and analysed by HPLC as described in Materials and methods. The calibration curve shown was obtained from three independent calibration curves.

 


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Fig. 3. Reverse phase HPLC-fluorescence profiles. (A) 100 µg calf thymus DNA were spiked with 30 pg/ml tetrol I-1, hydrolysed and neutralized as described above. (B) Tetrol I-1 formed after acid hydrolysis of 50 µg DNA isolated from HeLa cells after 2 h incubation with 50 nM (±)-anti-BPDE.

 
This high sensitivity allows the reproducible quantification of adduct formation and repair after incubation with low, non-cytotoxic concentrations of BPDE. To assess the adduct formation, A549 and HeLa cells were incubated with 1–100 nM (±)-anti-BPDE for 2 h (Figure 4Go). 1 nM (±)-anti-BPDE induced 9.4 ± 0.5 and 7.3 ± 0.5 adducts/108 base pairs in A549 and HeLa cells, respectively. In both cell lines a strong correlation between (±)-anti-BPDE concentration and adduct levels was observed. Interestingly A549 cells exerted up to 40% higher adduct levels compared with HeLa cells.



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Fig. 4. Formation of BPDE–DNA adducts induced by (±)-anti-BPDE in HeLa and A549 cells. Cells were incubated with (±)-anti-BPDE for 2 h. Adduct levels were quantified with HPLC/FD as described in Materials and methods. Shown are mean values of at least three independent determinations ± SD.

 
To investigate the repair of the induced DNA lesions, A549 cells were incubated with 5–100 nM (±)-anti-BPDE for 2 h, postincubated for 24 or 48 h and the remaining DNA damage was quantified. A549 cells exerted an efficient repair capacity within the first 24 h; however, at all concentrations applied between 20 and 35% of induced adducts could still be detected 48 h after treatment (Figure 5Go).



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Fig. 5. Repair of BPDE–DNA adducts in A549 cells. Logarithmically growing cells were incubated with (±)-anti-BPDE for 2 h. After 24 and 48 h of repair, respectively, adduct quantification was done as described in Materials and methods. Shown are mean values of at least three independent determinations ± SD.

 
Effects of soluble NiCl2and particulate black NiO on BPDE-induced DNA damage and cytotoxicity
To investigate the effects of soluble NiCl2 and particulate black NiO, different end points have been applied: (a) the cytotoxicity in A549 cells; (b) the effect on BPDE–DNA adduct formation; (c) the effect on the repair of the induced BPDE–DNA adducts; and (d) the effect on BPDE-induced cytotoxicity.

Cytotoxicity of NiCl2 and NiO
One important prerequisite to elucidate the effect of the different nickel compounds on (+)-anti-BPDE-induced DNA adduct formation and repair is the intracellular bioavailability of nickel(II). Cells were incubated for 20 and 24 h with NiCl2 and black NiO, respectively, since these incubation times have been shown to insure maximum intracellular nickel concentrations in case of NiCl2 (9) and significant uptake in case of NiO (12). The cytotoxicity of NiCl2 after 20 h of incubation is shown in Figure 6AGo. The colony-forming ability was only slightly reduced at concentrations up to 500 µM to ~70% and dropped thereafter, leaving ~30% of the cells viable after incubation with 1000 µM NiCl2. While after 24 h concentrations up to 0.5 µg/cm2 black NiO led to no reduction in colony-forming ability, 1.0 and 2.0 µg/cm2 NiO decreased colony-forming ability to ~78 and 49%, respectively (Figure 6BGo).




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Fig. 6. Cytotoxicity of NiCl2 (A) or NiO (B) and their effects on the repair of BPDE–DNA adducts in A549 cells. (A) Logarithmically growing cells were preincubated with NiCl2 for 18 h, coincubated with NiCl2 and 50 nM (+)-anti-BPDE for 2 h and postincubated for 6 h in the presence of NiCl2. (B) Logarithmically growing cells were preincubated with black NiO for 22 h, coincubated with NiO and 50 nM (+)-anti-BPDE for 2 h and postincubated for 8 h in the presence of NiO. 100% refer to the repair capacity in the absence of NiCl2 or NiO. The data represent mean values of at least four independent determinations + SD. For determination of cytotoxicity, logarithmically growing cells were treated with NiCl2 for 20 h or with NiO for 24 h, trypsinized and reseeded for colony-forming ability. The data represent mean values of 12 determinations ± SD. If error bars are not visible, they are smaller than the size of symbols.

 
Interaction with BPDE-induced DNA adduct formation
In a next step, we examined the effects of NiCl2 and NiO on BPDE-induced DNA adduct formation. A549 cells were preincubated with NiCl2 (18 h) or black NiO (22 h) and coincubated with 50 nM (+)-anti-BPDE for 2 h. The results are shown in Figure 7A and BGo: whereas NiCl2 showed no significant effect, black NiO reduced the adduct formation in a dose dependent manner. At the highest concentration of 2 µg/cm2 NiO adduct formation was decreased to ~69%.




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Fig. 7. Effect of soluble NiCl2 (A) or NiO (B) on BPDE–DNA adduct formation in A549 cells. Logarithmically growing cells were preincubated with NiCl2 for 18 h or with NiO for 22 h and coincubated with 50 nM (+)-anti-BPDE for 2 h. 100% refer to adduct levels in the absence of the respective nickel compound. The data represent mean values of at least four independent determinations ± SD in case of NiCl2 and at least six independent determinations ± SD in case of NiO.

 
Interaction with the repair of BPDE-induced DNA adducts
As described above, NiCl2 has been previously shown to interact with NER of UVC-induced DNA lesions at concentrations where the viability of the cells is not affected (9). Nevertheless, no such data are available for particulate nickel compounds. Furthermore, with respect to NiCl2, it is still unclear whether repair inhibition also applies to other types of DNA damage removed by NER. To elucidate the effects of both soluble NiCl2 and NiO on the removal of BPDE-induced DNA adducts, A549 cells were preincubated with NiCl2 (18 h) or NiO (22 h), subsequently coincubated with 50 nM (+)-anti-BPDE for 2 h and postincubated in the presence of the respective nickel compound for 6 or 8 h. The remaining DNA lesions were quantified by HPLC/fluorescence assay as described in Materials and methods. Out of 400 lesions/108 base pairs induced by this treatment, ~40% were repaired within this time period in the absence of nickel. However, both soluble NiCl2 and particulate black NiO exerted a dose dependent repair inhibition at low, non-cytotoxic concentrations (Figure 6A and BGo). Thus, 100 µM NiCl2 led to a pronounced repair inhibition; after 500 µM the repair was reduced to ~36%. In the presence of black NiO there was an even more severe decrease in repair activity, yielding only ~35 and 15% residual repair capacity at doses of 0.5 and 2.0 µg/cm2 NiO, respectively, compared with cells treated with (+)-anti-BPDE in the absence of NiO.

Effect of NiCl2and NiO on BPDE-induced cytotoxicity
The cytotoxicity of (+)-anti-BPDE in the presence and absence of NiCl2 and black NiO has been examined by determination of the colony-forming ability. A549 cells were preincubated with NiCl2 (18 h) or black NiO (22 h), coincubated with (+)-anti-BPDE for 2 h, and postincubated in the presence of the corresponding nickel compound. The effects of NiCl2 and black NiO on (+)-anti-BPDE-induced cytotoxicity are shown in Figure 8Go. Regarding (+)-anti-BPDE alone, the colony-forming ability was only slightly decreased at concentrations up to 50 nM; thereafter it dropped to ~60% at 100 nM. In the presence of 250 or 500 µM NiCl2, however, the colony-forming ability was reduced markedly, yielding pronounced cytotoxicity at 50 nM and 5 nM (+)-anti-BPDE, respectively (Figure 8AGo). In case of NiO an increase in BPDE-induced cytotoxicity was only seen at 100 nM (+)-anti-BPDE (Figure 8BGo).




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Fig. 8. Effect of NiCl2 (A) or NiO (B) on (+)-anti-BPDE-induced cytotoxicity. (A) Logarithmically growing A549 cells were preincubated with NiCl2 for 18 h, coincubated with NiCl2 and (+)-anti-BPDE for 2 h, postincubated with NiCl2 for 6 h, trypsinized and reseeded for colony-forming ability. 100% refer to solvent controls or to NiCl2-treated cells, respectively. (B) Logarithmically growing A549 cells were preincubated with NiO for 22 h, coincubated with NiO and (+)-anti-BPDE for 2 h, postincubated in the presence of NiO for 6 h, trypsinized and reseeded for colony-forming ability. 100% refer to solvent controls or to NiO-treated cells, respectively. The data represent mean values of at least three determinations ± SD. If error bars are not visible, they are smaller than the size of symbols.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
The results presented in this study demonstrate a DNA repair inhibition of B[a]P-induced stable DNA adducts by water-soluble NiCl2 as well as by largely water-insoluble black NiO particles.

One important prerequisite to study the effect of different nickel compounds on the B[a]P-induced DNA adduct formation and repair was the sensitive quantification of the DNA lesions. This was achieved by the HPLC/fluorescence procedure described previously (29,30) with modifications. In the present study, it allowed the quantification of as little as 1 adduct/108 base pairs or ~60 adducts per cell, requiring 10–100 µg DNA; the small standard deviations derived from independent experiments demonstrate the high reproducibility of the method. It was therefore possible to measure adduct formation and repair on non-cytotoxic conditions. In general, this approach is sensitive enough to determine B[a]P-induced adduct levels in human lymphocytes in the occupationally not exposed general population (30,31).

Concerning the effect of nickel compounds on the extent of BPDE-induced DNA adducts, a dose-dependent reduction to ~70% was observed in case of NiO, while NiCl2 did not influence adduct formation. Several reasons could account for this observation. First, with respect to usually higher concentrations of Ni(II) ions in the nucleus obtained with particulate nickel compounds compared with water-soluble nickel compounds, nickel binding to DNA bases could prevent adduct formation. Nevertheless, Ni(II) exerts much higher affinities to amino acids in proteins and glutathione compared with DNA bases, resulting in very low Ni-DNA binding on cellular conditions (32). Other explanations could be the interaction of nickel particles with hydrolysis, uptake and intracellular distribution of BPDE as well as interactions with BPDE metabolism. For example, BPDE could associate with NiO extracellularly, resulting in decreased uptake and increased extracellular hydrolysis. Nevertheless, the exact reason for decreased adduct formation is not known and interpretations remain speculative at this point. Similarly, there was a pronounced difference in adduct formation in both cell lines, with A549 showing higher adduct levels, which may be due to differences in BPDE uptake and/or detoxification.

Concerning the repair of BPDE-induced DNA adducts, most of the lesions are removed within 24 h. Nevertheless, even 48 h after treatment, between 20 and 35% of the initial adduct frequency still remained, corresponding to ~120 adducts per 108 base pairs after incubation with 100 nM BPDE. This is due to incomplete lesion removal by NER mediating the repair of stable BPDE–DNA adducts. NER removes structurally unrelated bulky base adducts generating significant helical distortions. According to current knowledge it involves at least 30 different proteins and enzymes in mammalian cells, including those which are defective in patients suffering from the DNA repair disorder xeroderma pigmentosum complementation groups A through G (33). Two different pathways can be discriminated: the global genome repair (GG-NER) operating in all parts of the genome and the transcription-coupled repair (TC-NER) eliminating DNA damage from the transcribed strand of active genes. While TC-NER is usually fast and efficient to restore transcription, GG-NER is usually slower and may be incomplete, leading to an accumulation of mutations in poorly repaired regions (34,35). Accordingly, three levels of repair efficiency have been identified in human fibroblasts after treatment with BPDE. Thus, the transcribed strand of the active HPRT gene was repaired about twice as fast compared with the non-transcribed strand, resulting in 53% and 26% of adduct removal after 7 h, respectively. In contrast, only 14% of BPDE adducts were lost from an inactive locus 754 within 20 h (36). The longevity of at least some adducts was also shown in humans: When comparing PAH adduct levels in lung autopsy samples of non-smokers, ex-smokers and smokers, lowest frequencies were found in the first group, intermediate frequencies in the second and highest values in the third group. Nevertheless, almost all samples even of the non-smoking group had detectable PAH-induced DNA lesions, indicating that even low environmental exposure leads to unrepaired DNA adducts. Furthermore, very similar levels were found in smokers and ex-smokers, indicating that the lesions are stable and persist for a long time after cessation of smoking (37).

Concerning the effect of nickel compounds on the repair of BPDE-induced DNA adducts, both soluble NiCl2 and particulate black NiO impaired the removal of BPDE–DNA adducts considerably. NiCl2 reduced the repair in a dose-dependent manner down to 36% compared with BPDE alone on conditions where the colony-forming ability was reduced only slightly to 70% of control. The results support previous findings, where the removal of UV- and platinum-induced DNA lesions were inhibited in HeLa cells (9,37) and point towards an overall inhibition of NER. For NiO a repair inhibition was demonstrated for the first time. When compared with NiCl2, it exerted an even more pronounced effect on repair capacity; thus, on completely non-cytotoxic conditions with respect to colony-forming ability, lesion removal was reduced to ~35% of control. This may be due to higher concentrations reaching the nucleus after phagocytosis of particles (4,12). Regarding these findings, repair inhibition by nickel(II) appears to be independent of the compound applied, and the results as such do not provide an explanation for the marked differences in carcinogenic potencies between water-soluble and particulate nickel compounds described above. However, when considering the carcinogenicity in humans and experimental animals, the retention times in the body have to be taken into account. Thus, analysis of nickel contents in rat lungs after inhalation of different nickel compounds revealed especially for nickel oxide an impaired clearance and up to 1000-fold higher persistent nickel lung burdens compared with water-soluble nickel sulfate (3). Therefore, exposure to particulate nickel compounds may give rise to continuous DNA repair impairment and thus the biological consequences may be far more severe. It cannot be excluded, however, that due to higher intranuclear nickel concentrations provoked by particulate nickel compounds additional genotoxic and epigenetic events like increased oxidant concentrations and/or altered gene expression contribute to their high carcinogenicity.

With respect to BPDE-induced DNA adducts, the diminished repair provides a plausible explanation for early results demonstrating a comutagenic effect of NiSO4 with benzo[a]pyrene in primary cultures of Syrian hamster embryos (39). Our results seem to contradict, however, more recent observations by Hamdan et al. (40), where Ni3S2 was protective towards BPDE-induced mutagenicity in human fibroblasts. Nevertheless, these authors used conditions where the colony-forming ability after combined exposure was reduced considerably down to 2%, which do not seem to be adequate to measure mutagenicity at the hprt locus based on mutant colony recovery.

The results presented in this study provide evidence that even on non-cytotoxic conditions DNA lesions induced by BPDE and thus also by B[a]P persist for prolonged periods of time; since the level of stable adducts induced by PAH correlates with the carcinogenic potency (16), both nickel compounds may increase the risk of B[a]P-induced tumor formation. This is relevant not only for occupational conditions where combined exposures occur frequently, but also for environmental exposure. For example, both PAH including B[a]P and nickel compounds are frequently associated in ambient air particles such as coal fly ash (13), and thus DNA repair inhibition has to be taken into account for risk assessment. Since DNA is damaged permanently by exogenous and endogenous mutagens, and genetic integrity depends largely on efficient repair, our results provide further evidence that DNA repair inhibition may be one predominant mechanism in nickel-induced carcinogenicity.


    Notes
 
3 To whom reprint requests should be sent: University of Karlsruhe, Department of Food Chemistry and Toxicology, Postfach 6980, D-76128 Karlsruhe, Germany Email: Andrea.Hartwig{at}chemie.uni-karlsruhe.de Back


    Acknowledgments
 
The authors thank Dr Adriana Oller, NIPERA, Durham, North Carolina, USA, for the kind gift of NiO particles and Mrs Christiane Glaser, Universität Karlsruhe, Institut für Werkstoffe der Elektrotechnik, for scanning electron microscopy of NiO particles. This work was supported by BWPLUS, grant No. BW BG 99012, and by the Deutsche Forschungsgemeinschaft, grant no. Ha 2372/1-2.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 26, 2001; revised September 17, 2001; accepted October 2, 2001.