Carcinogenesis vol.23 no.1 pp.47–53, 2002
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
1Institut
für Lebensmittelchemie und Toxikologie, Universität Karlsruhe,
D-76128 Karlsruhe and 2Biochemisches Institut für Umweltcarcinogene,
Prof Dr G.Grimmer-Stiftung, D-22927 Grosshansdorf, Germany
3To
whom reprint requests should be sent: University of Karlsruhe,
Department of Food Chemistry and Toxicology, Postfach 6980, D-76128
Karlsruhe, Germany
Email: [email protected]
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 9,10epoxide (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.
Introduction
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
Abbreviations: B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene-7,8-diol 9,10epoxide; 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-7,8,9,10tetrahydrobenzo[a]pyrene; THF, tetrahydrofuran.
© Oxford University Press
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
47
T.Schwerdtle, A.Seidel and A.Hartwig
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
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 ⫻ 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, 100 U/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.
48
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 ⫻ 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 precolumn (Luna C18, 5 µm, 4.6 ⫻ 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 ⫻ 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
Cytotoxicity of BPDE
The cytotoxicity of (⫾)-anti-BPDE was determined by colonyforming ability after 2 h incubation (Figure 1). 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.
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 2). 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
Effect of nickel on benzo[a]pyrene DNA adduct repair
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.
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.
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.
in Figure 3. 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.
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
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.
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.
1–100 nM (⫾)-anti-BPDE for 2 h (Figure 4). 1 nM (⫾)-antiBPDE 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.
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 5).
Effects of soluble NiCl2 and particulate black NiO on BPDEinduced 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.
49
T.Schwerdtle, A.Seidel and A.Hartwig
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.
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 6A. 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 6B).
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 B: whereas NiCl2 showed no
50
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.
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%.
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 B). 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
Effect of nickel on benzo[a]pyrene DNA adduct repair
cytotoxicity at 50 nM and 5 nM (⫹)-anti-BPDE, respectively
(Figure 8A). In case of NiO an increase in BPDE-induced
cytotoxicity was only seen at 100 nM (⫹)-anti-BPDE
(Figure 8B).
Discussion
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 colonyforming 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.
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 NiCl2 and 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 8. Regarding (⫹)-anti-BPDE alone, the colonyforming 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 colonyforming ability was reduced markedly, yielding pronounced
The results presented in this study demonstrate a DNA repair
inhibition of B[a]P-induced stable DNA adducts by watersoluble 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 transcriptioncoupled repair (TC-NER) eliminating DNA damage from the
51
T.Schwerdtle, A.Seidel and A.Hartwig
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 nonsmokers, 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 PAHinduced 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 dosedependent 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 watersoluble 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
52
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.
Acknowledgements
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
1. IARC (1990) Monographs on the Evaluation of the Carcinogenic Risks to
Humans, Vol. 49, Chromium, Nickel and Welding. IARC, Lyon.
2. Oller,A.R., Costa,M. and Oberdorster,G. (1997) Carcinogenicity assessment
of selected nickel compounds. Toxicol. Appl. Pharmacol., 143, 152–166.
3. Dunnick,J.K., Elwell,M.R., Radovsky,A.E., Benson,J.M., Hahn,F.F.,
Nikula,K.J., Barr,E.B. and Hobbs,C.H. (1995) Comparative carcinogenic
effects of nickel subsulfide, nickel oxide, or nickel sulfate hexahydrate
chronic exposures in the lung. Cancer Res., 55, 5251–5256.
4. Costa,M., Simmons-Hansen,J., Bedrossian,C.W., Bonura,J. and
Caprioli,R.M. (1981) Phagocytosis, cellular distribution and carcinogenic
activity of particulate nickel compounds in tissue culture. Cancer Res.,
41, 2868–2876.
5. Kasprzak,K.S. (1995) Possible role of oxidative damage in metal-induced
carcinogenesis. Cancer Invest., 13, 411–430.
6. Bal,W., Kozlowski,H. and Kasprzak,K.S. (2000) Molecular models in
nickel carcinogenesis. J. Inorg. Biochem., 79, 213–218.
7. Lee,Y.W., Klein,C.B., Kargacin,B., Salnikow,K., Kitahara,J., Dowjat,K.,
Zhitkovich,A., Christie,N.T. and Costa,M. (1995) Carcinogenic nickel
silences gene expression by chromatin condensation and DNA methylation:
a new model for epigenetic carcinogens. Mol. Cell Biol., 15, 2547–2557.
8. Hartwig,A. and Beyersmann,D. (1989) Enhancement of UV-induced
mutagenesis and sister-chromatid exchanges by nickel ions in V79 cells:
evidence for inhibition of DNA repair. Mutat. Res., 217, 65–73.
9. Hartwig,A., Mullenders,L.H., Schlepegrell,R., Kasten,U. and Beyersmann,
D. (1994) Nickel (II) interferes with the incision step in nucleotide excision
repair in mammalian cells. Cancer Res., 54, 4045–4051.
10. Hartmann,M. and Hartwig,A. (1998) Disturbance of DNA damage
recognition after UV-irradiation by nickel (II) and cadmium (II) in
mammalian cells. Carcinogenesis, 19, 617–621.
11. 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.
12. Fletcher,G.G., Rossetto,F.E., Turnbull,J.D. and Nieboer,E. (1994) Toxicity,
uptake and mutagenicity of particulate and soluble nickel compounds.
Environ. Health Perspect., 102 Suppl. 3, 69–79.
13. Borm,P.J. (1997) Toxicity and occupational health hazards of coal fly ash
(CFA). A review of data and comparison to coal mine dust. Ann. Occup.
Hyg., 41, 659–676.
Effect of nickel on benzo[a]pyrene DNA adduct repair
14. Drouin,E.E. and Loechler,E.L. (1993) AP sites are not significantly
involved in mutagenesis by the (⫹)-anti-diolepoxide of benzo[a]pyrene:
the complexity of its mutagenic specificity is likely to arise from adduct
conformational polymorphism. Biochemistry, 32, 6555–6562.
15. Hall,M., Grover,P.L. (1990) Polycyclic aromatic hydrocarbons: metabolism,
activation and tumor initiation. In Cooper,C.S. and Grover,P.L. (eds)
Chemical Carcinogenesis and Mutagenesis I. Springer Verlag, Berlin,
pp. 327–372.
16. Melendez-Colon,V.J., Luch,A., Seidel,A. and Baird,W.M. (1999) Cancer
initiation by polycyclic aromatic hydrocarbons results from formation
of stable DNA adducts rather than apurinic sites. Carcinogenesis, 20,
1885–1891.
17. Bolton,J.L., Trush,M.A., Penning,T.M., Dryhurst,G. and Monks,T.J. (2000)
Role of quinones in toxicology. Chem. Res. Toxicol., 13, 135–160.
18. Lieber,M., Smith,B., Szakal,A., Nelson-Rees,W. and Todaro,G. (1976) A
continuous tumor-cell line from a human lung carcinoma with properties
of type II alveolar epithelial cells. Int. J. Cancer, 17, 62–70.
19. Foster,K.A., Oster,C.G., Mayer,M.M., Avery,M.L. and Audus,K.L. (1998)
Characterization of the A549 cell line as a type II pulmonary epithelial
cell model for drug metabolism. Exp. Cell Res., 243, 359–366.
20. Urani,C., Doldi,M., Crippa,S. and Camatini,M. (1998) Human-derived cell
lines to study xenobiotic metabolism. Chemosphere, 37, 2785–2795.
21. Yagi,H., Thakker,D.R., Hernandez,O., Koreeda,M. and Jerina,D.M. (1977)
Synthesis and reactions of the highly mutagenic 7,8-diol 9,10-epoxides of
the carcinogen benzo[a]pyrene. J. Am. Chem. Soc., 99, 1604–1611.
22. McCaustland,D.J. and Engel,J.F. (1975) Metabolites of polycyclic aromatic
hydrocarbons. II. Synthesis of 7,8-dihydrobenzo[a]pyrene-7,8-diol and
7,8-dihydrobenzo[a]pyrene-7,8-epoxide. Tetrahedron Lett., 2549–2552.
23. Fu,P.P. and Harvey,R.G. (1977) Synthesis of the diols and diolepoxides of
carcinogenic hydrocarbons. Tetrahedron Lett., 2059–2062.
24. Yang,S.K., Gelboin,H.V., Weber,J.D., Sankaran,V., Fischer,D.L. and
Engel,J.F. (1977) Resolution of optical isomers by high-pressure liquid
chromatography. The separation of benzo[a]pyrene trans-diol derivatives.
Anal. Biochem., 78, 520–526.
25. Harvey,R.G. and Cho,H. (1977) Efficient resolution of the dihydrodiol
derivatives of benzo[a]pyrene by high-pressure liquid chromatography of
the related (–)-dimenthoxyacetates. Anal. Biochem., 80, 540–546.
26. Yagi,H., Vyas,K.P., Tada,M., Thakker,D.R. and Jerina,D.M. (1982) Synthesis
of the enantiomeric bay-region diol epoxides of benz[a]anthracene and
chrysene. J. Org. Chem., 47, 1110–1117.
27. Yagi,H., Akagi,H., Thakker,D.R., Mah,H.D., Koreeda,M. and Jerina,D.M.
(1977) Absolute sterochemistry of the highly mutagenic 7,8-diol 9,10epoxides derived from the potent carcinogen trans-7,8-dihydroxy-7,8dihydrobenzo[a]pyrene. J. Am. Chem. Soc., 99, 2358–2359.
28. Sambrock,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular cloning, 2nd
edn. Cold Spring Harbor, Laboratory Press, NY.
29. Alexandrov,K., Rojas,M., Geneste,O., Castegnaro,M., Camus,A.M.,
Petruzzelli,S., Giuntini,C. and Bartsch,H. (1992) An improved fluorometric
assay for dosimetry of benzo[a]pyrene diol-epoxide-DNA adducts in
smokers’ lung: comparisons with total bulky adducts and aryl hydrocarbon
hydroxylase activity. Cancer Res., 52, 6248–6253.
30. Rojas,M., Alexandrov,K., van Schooten,F.J., Hillebrand,M., Kriek,E. and
Bartsch,H. (1994) Validation of a new fluorometric assay for
benzo[a]pyrene diolepoxide-DNA adducts in human white blood cells:
comparisons with 32P- postlabeling and ELISA. Carcinogenesis, 15,
557–560.
31. Pavanello,S., Favretto,D., Brugnone,F., Mastrangelo,G., Dal Pra,G. and
Clonfero,E. (1999) HPLC/fluorescence determination of anti-BPDE–DNA
adducts in mononuclear white blood cells from PAH-exposed humans.
Carcinogenesis, 20, 431–435.
32. Ciccarelli,R.B. and Wetterhahn,K.E. (1984) Nickel-bound chromatin,
nucleic acids and nuclear proteins from kidney and liver of rats treated
with nickel carbonate in vivo. Cancer Res., 44, 3892–3897.
33. de Boer,J. and Hoeijmakers,J.H. (2000) Nucleotide excision repair and
human syndromes. Carcinogenesis, 21, 453–460.
34. Mullenders,L.H., Vrieling,H., Venema,J. and van Zeeland,A.A. (1991)
Hierarchies of DNA repair in mammalian cells: biological consequences.
Mutat Res., 250, 223–228.
35. Leadon,S.A. (1999) Transcription-coupled repair of DNA damage:
unanticipated players, unexpected complexities. Am. J. Hum. Genet., 64,
1259–1263.
36. Chen,R.H., Maher,V.M., Brouwer,J., van de Putte,P. and McCormick,J.J.
(1992) Preferential repair and strand-specific repair of benzo[a]pyrene diol
epoxide adducts in the HPRT gene of diploid human fibroblasts. Proc.
Natl Acad. Sci. USA, 89, 5413–5417.
37. Lodovici,M., Akpan,V., Giovannini,L., Migliani,F. and Dolara,P. (1998)
Benzo[a]pyrene diol-epoxide DNA adducts and levels of polycyclic
aromatic hydrocarbons in autoptic samples from human lungs. Chem. Biol.
Interact., 116, 199–212.
38. Krueger,I., Mullenders,L.H. and Hartwig,A. (1999) Nickel (II) increases
the sensitivity of V79 Chinese hamster cells towards cisplatin and
transplatin by interference with distinct steps of DNA repair.
Carcinogenesis, 20, 1177–1184.
39. Rivedal,E. and Sanner,T. (1980) Synergistic effect on morphological
transformation of hamster embryo cells by nickel sulphate and
benzo[a]pyrene. Cancer Lett., 8, 203–208.
40. Hamdan,S., Morse,B. and Reinhold,D. (1999) Nickel subsulfide is similar
to potassium dichromate in protecting normal human fibroblasts from the
mutagenic effects of benzo[a]pyrene diolepoxide. Environ. Mol. Mutagen.,
33, 211–218.
Received July 26, 2001; revised September 17, 2001; accepted October 2, 2001
53