TOXICOLOGJCAL SCIENCES 46, 2 6 0 - 2 6 5 (1998)
ARTICLE NO. TX982512
Sensitive Quantitation of Chromium-DNA Adducts by Inductively
Coupled Plasma Mass Spectrometry with a Direct Injection
High-Efficiency Nebulizer
Jatinder Singh,*1 John A. McLean,f' Daryl E. Pritchard,* Akbar Montaser,f2 and Steven R. Patierno*'2
* Department of Pharmacology, and ^Department of Chemistry, The George Washington University, Washington, DC 20052
Received January 16, 1998; accepted May 29, 1998
tory tract ulcerations in occupationally exposed workers
Sensitive Quantitation of Chromium-DNA Adducts by Induc- (IARC, 1990). Chromium enters cells in the form of Cr(VI)
tively Coupled Plasma Mass Spectrometry with a Direct Injection oxyanions and is reduced intracellularly by several reducing
High-Efficiency Nebulizer. Singh, J., McLean, J. A., Pritchard, D., agents, such as ascorbate and glutathione, to the predominant
Montaser, A., and Patierno, S. R. (1998). Toxicol. Sci. 46, 260-265.
trivalent species, Cr(III), which forms stable Cr(III)-DNA
A novel method is described for the sensitive detection of chro- (Cr-DNA) monoadducts (Snow, 1992). Previous studies of Cr
mium-DNA adducts. Chromium-DNA adducts were determined exposure have been limited to using radioactive 5l Cr in culin 1 pig of DNA from normal human lung fibroblasts exposed to tured cells (Xu et ah, 1994) or measuring its atomic absorption
sodium chromate using microscale flow injection analysis with a in tissue extracts and body fluids (Finley et ah, 1997; De Flora
direct injection high-efficiency nebulizer and inductively coupled
5l
plasma mass spectrometry detection. The frequency of Cr-DNA et ah, 1997). The use of Cr is inconvenient and potentially
adducts increased in a dose-dependent sigmoidal manner, indicat- hazardous and obviously cannot be used to measure Cr-DNA
ing saturation and toxicity. The low detection limits (on the order adducts in humans exposed to nonradioactive isotopes of chroof parts per trillion) allows the detection of as few as two Cr mium. Atomic absorption measurement requires a large sample
adducts per 10,000 bases, which, coupled with the small DNA size and thus is generally limited to measuring Cr in blood or
sample requirement, makes this technique suitable for measuring urine. Furthermore, the atomic absorption quantitation of chrometal-DNA adducts as biomarkers of exposure to toxic and car- mium in body fluids does not reflect the bioavailable fraction of
cinogenic metals such as Cr, in cultured cells, animals, and Cr that is taken up by the cells and is capable of causing
humans.
© 1998 Society of Toxicology.
cytotoxicity and genotoxicity. In fact, total Cr in the blood is
likely to be predominantly in the reduced Cr(III) form which is
not bioavailable for cellular uptake. Additionally, a large fracCertain compounds of transition metals such as Cr, Ni, Cd, tion of the Cr in the blood may not be "free" because it may be
Pb, Hg, and Cu are toxic, mutagenic, and carcinogenic (re- bound to serum proteins such as globulins. Another confoundviewed by Snow, 1992; Goyer and Cherian, 1995). DNA is ing factor is that Cr concentrations in body fluids are affected
targeted by metal ions that enter the cells, and metal-DNA by diet. For example, beer, due to brewer's yeast, contains Cr
adducts are mechanistically related to the toxicity, mutagenic- that is excreted via the urine and is a confounding variable
ity, and carcinogenicity of these metals. Therefore, metal- associated with urinary assessment of human exposure to Cr
DNA adducts constitute a toxicologically salient biomarker for (Bukowski et ah, 1991).
Chromium-induced DNA-protein crosslinks (DPC), dethe assessment of environmental or occupational exposure to
these metals. Furthermore, quantitation of metal-DNA adducts tected by potassium chloride-sodium dodecyl sulfate-mediated
may be applicable in risk assessment studies of metal carcino- precipitation, have also been used as an indirect index of Cr
genesis. Thus, for routine analysis, a highly sensitive technique exposure in both cultured cells and mice (Zhitkovich and
Costa, 1992) and, to some extent, in Cr-exposed industrial
requiring a small DNA sample size is desirable.
We have been investigating the molecular and cellular ef- workers such as welders (Costa et ah, 1993). This technique
fects of Cr, a prototypical toxic and carcinogenic metal. Certain has a number of limitations, including being rather tedious and
hexavalent compounds of Cr are environmental contaminants subject to considerable interindividual and interlaboratory vari(Freeman and Lioy, 1997) and cause lung cancer and respira- ation (Costa et ah, 1996). The nature of the crosslinks measured by this technique is also not clear. For example, it has
been
reported that the predominant type of DPC formed in
1
These authors have contributed to an equal extent.
2
chromium-treated cells is between DNA and glutathione or
This work represents an equal collaboration between these two principal
amino acids such as cysteine and histidine (Zhitkovich et ah,
investigators. Reprint requests may be addressed to either author.
1096-6080/98 S25.00
Copyright © 1998 by the Society of Toxicology.
All rights of reproduction in any form reserved.
26
°
261
QUANTITATION OF Cr-DNA ADDUCTS BY 1CPMS
1995), molecules that are probably too small to mediate the
precipitation of crosslinked DNA. It has also been reported that
as much as half of the DPC formed in Cr-treated cells does not
contain a Cr atom and may be mediated by oxidative mechanisms (Mattagajasingh and Misra, 1996), rather than by coordinate crosslinking. Additionally, it appears that most of the
DPC in cultured cells measured by this technique occurs predominantly at markedly cytotoxic doses of hexavalent chromate (Costa et al, 1996; Miller and Costa, 1990), suggesting
that this procedure may not be amenable to environmental
monitoring unless a person has sustained a highly toxic exposure to chromium. Indeed, there was no significant increase in
DPC, as measured by this method, in human leukocytes following acute ingestion of nontoxic amounts of Cr in drinking
water (Kuykendall et al., 1997). Finally, DPC can be caused by
a number of environmental agents besides Cr (Zhitkovich and
Costa, 1992); thus the specificity of this technique for monitoring Cr exposure is limited.
Clearly, there is a need to develop a more sensitive, selective, direct, and toxicologically relevant method of measuring
cellular exposure and uptake of bioavailable chromium. We
have developed a highly sensitive and specific technique for
the measurement of Cr-DNA adducts by inductively coupled
plasma mass spectrometry (ICPMS). ICPMS is the most powerful technique for trace and ultratrace elemental analysis
(Montaser, 1998). However until recently, sample introduction
into the ICPMS was performed with conventional nebulizerspray chamber combinations typically consuming 1-3 ml/min
of sample solution and providing only 1-5% analyte transport
efficiency. These conditions, while suitable for analyzing environmental samples (e.g., river water) and body fluids (e.g.,
urine), precluded the analysis of microliter volumes of nucleic
acids samples. Here we report on the development and application of an ICPMS technique to detect Cr-DNA adducts
formed in the DNA of cultured normal human lung fibroblasts
(HLF) exposed to sodium chromate. A direct injection highefficiency nebulizer (DIHEN) coupled with microscale flow
injection analysis (/xFIA) was used for the introduction of
microliter volumes of Cr-DNA into the ICPMS. HLF were
used as the cellular model because of the association between
inhaled chromium, lung cancer and respiratory ulcerations
(I ARC, 1990).
The DIHEN (McLean et al, 1998) is particularly well suited
for this analysis, because it provides: (1) 100% analyte transport
efficiency, (2) high sensitivity at solution flow rates of 1-100
/x-l/min, (3) improved precision in the absence of a spray chamber,
(4) reduced sample dispersion, and (5) parts per trillion (ppt)
powers of detection similar to, or better than, those provided by
conventional sample introduction systems operated at 1 ml/min.
The DIHEN is also a simple, low-cost, micronebulizer compared
to other devices (Montaser et al, 1998). Previously, ICPMS was
used to study the interaction of Cd, Cu, Ag, and Pb with the
nucleosomes of cultured rat hepatocytes (Denizeau and Marion,
1989), and to evaluate the DNA-binding efficacy of platinum-
TABLE 1
Operating Conditions for the Ar ICPMS Instrument and
Microscale Flow Injection System
ICPMS System
RF power, W
Nominal frequency, MHz
RF generator type
Induction coil circuitry
Sampling depth (above load coil), mm
Sampler (orifice diameter, mm)
Skimmer (orifice diameter, mm)
Outer gas flow rate, liters/min
Intermediate gas flow rate, liters/min
Sample introduction system
Carrier solution flow rate, /xl/min
Injector gas flow rate, liters/min
Sample loop volume, /u.1
Data acquisition parameters
Scan mode
Points/mass
Resolution, amu
Sweeps/reading
Readings/replicate
Replicates
Dwell time/mass, ms
Integration time, ms
PE-Sciex Elan 6000
1500
40
Free-running
3-turn coil, Plasmalok
11
Nickel, 1.1
Nickel, 0.9
15
1.2
DIHEN, see text
85
0.25
20
Peak hopping
1
0.7
15
1
500
20
300
based anticancer agents such as cisplatin and carboplatin in vivo
(Bonetti et al, 1996). However, these studies cannot be extended
to a routine assay for DNA-bound metals due to the large sample
size requirements.
The modified ICPMS technique was able to quantitate the
dose-dependent formation of Cr-DNA adducts in the DNA of
HLF. These results suggest that the proposed methodology can
be potentially useful for metal toxicology, risk assessment, and
human biomonitoring studies.
METHODS AND MATERIALS
The direct injection high-efficiency nebulizer. Recently, the DIHEN3 was
introduced as a micronebulizer for ICPMS and is discussed elsewhere
(McLean et al, 1998). Sample and carrier solutions were delivered to the
DIHEN via a four-channel peristaltic pump.4 To reduce pressure pulsation
induced noise in the system, narrow bore tygon tubing5 (0.015 in. i.d.) was
used for solution flow rates of 85 /xl/min. The injector gas flow rate was
controlled by a mass flow controller6 to maintain an injector gas flow rate of
0.25 liters/min.
The argon inductively coupled plasma mass spectrometer instrument. In
all cases, metal concentrations were determined using an Elan 6000 ICPMS
system7 with the operating conditions and data acquisition parameters listed in
Table 1. Data were collected in the peak hopping mode, and the lens voltage
was auto-optimized for each m/z.
3
4
5
6
7
J E Meinhard Associates Inc., Santa Ana, CA.
Model Rabbit, Rainin Instrument Co Inc., Wobum, MA.
Astoria-Pacific Inc., Clackamas, OR.
Model 8200, Matheson Gas Products, East Rutherford, NJ.
Perkin-Elmer/Sciex Corporation, Norwalk, CT.
262
SINGH ET AL.
Microscale flow injection-ICPMS.
For /J.FIA-DIHEN-ICPMS experiments, a computer actuated six-way flow injection valve8 was used with a
20-jxl sample loop. Sample was loaded into the sample loop and carrier
solution was delivered to the DIHEN at an uptake rate of 85 /il/min via the
aforementioned four-channel peristaltic pump. The dead volume from the
injection valve to the end of the nebulizer was reduced to <8.5 fil by inserting
a 260-mm length of 0.008-in. i.d. X 0.016-in. o.d. PTFE tubing9 directly into
the back of the DIHEN sample capillary to the point where the capillary tapers.
To connect this microbore tubing to the injection valve, a 0.020-in. i.d. X
1/16-in. o.d. FEP tubing sleeve10 was placed over PTFE tubing. The /xFIADIHEN-ICPMS system was optimized daily through operating the system in a
continuous flow mode. Peak heights and areas were determined by exporting
data files as signal intensity versus time in ASCII format. The data were then
analyzed using a commercial statistics package."
Reagents. A multielement solution containing 10 ng/ml of Cr, As, and Pb
was prepared by diluting 1000-/j.g/ml stock solutions12 with a 2% solution of
high-purity nitric acid13 and 18-Mfl-cm distilled deionized water (DDW). This
solution was used to determine reproducibility of the yuFIA-DIHEN-ICPMS
technique.
Chromium treatment of HLF and purification of chromium-bound DNA.
IX 24 HLF 14 cells were grown in T150 tissue culture flasks in Ham's F-12K15
medium supplemented with 15% fetal bovine serum16 in a 95% air and 5%
carbon dioxide humidified atmosphere at 37°C. Exponentially growing, subconfluent cells were treated with different concentrations of sodium chromate17
(in the medium) for 2 h. After the treatment, cells were detached by trypsinization and DNA was extracted by standard phenol-chloroform procedures
(Sambrook et al, 1989) and reconstituted in distilled, deionized water at a
concentration of 0.0255 Aig//xl. The background levels of Cr in the water were
approximately 60 parts per trillion. This was deducted when calculating
Cr-DNA adduct levels. The DNA solutions were then subjected to ICPMS
analysis as described above.
Cr
Cr
RESULTS
Signal stability for the p,FIA-DIHEN-ICPMS determination
of Pb, As, and Cr isotopes was evaluated for 10 repeated
injections of 200 pg of each element per injection. The peak
profiles for As (m/z = 75) and the major isotopes of Pb (m/z =
206, 207, 208) are shown in Fig. 1A, and the peak profiles for
two minor isotopes of Cr (m/z = 50, 53) are shown in Fig. IB.
Isobaric interference from 40 Ar l2 C precluded monitoring the
major isotope of chromium, namely, 52Cr which is 83.79%
naturally abundant (Lide, 1995). Although 75As is 100% naturally abundant, it exhibits relatively low signal intensity due
to a low degree of ionization (Horlick and Montaser, 1998),
because of its relatively high first ionization potential (9.81
eV). Peak-to-peak reproducibilities based on peak areas and
heights, and the percent natural abundance of these isotopes are
8
CETAC Technologies Inc., Omaha, NE.
Cole-Parmer, Vernon Hills, IL.
10
Upchurch Scientific, Oak Harbor, WA.
1
' Microcal Origin 3.73, Microcal Software Inc., Northampton, MA.
12
SPEX CertiPrep Inc., Metuchen, NJ.
13
Optima Grade, Fisher Scientific, Pittsburgh, PA.
14
ATCC, Rockville, MD.
15
Irvine Scientific, Santa Ana, CA.
16
HyClone, Logan, UT.
17
J. T. Baker Chemical Co., Philipsburg, NJ.
9
750
FIG. 1. (A) Peak profiles for 10 repeated injections of 200 pg lead and
arsenic. (B) Peak profiles for 10 repeated injections of 200 pg chromium.
shown in Table 2. Precision, defined as the percent relative
standard deviation (%RSD), ranged from 0.9 to 2.8 and 0.9 to
2.3% RSD (N = 10) based on peak areas and peak heights,
respectively.
The relative concentration of Cr in the Cr-DNA samples
was then determined with / A F I A - D I H E N - I C P M S monitoring
Cr at m/z = 53. Isobaric interference from 40Ar14N precluded
monitoring 54Cr. Additionally, isobaric interference from
34 16 32 I8
S 0, S O, and 33S17O species derived from protein, SDS,
and enzyme contamination precluded monitoring 50Cr. These
interferences may be reduced, or eliminated, through using
cool plasma techniques, helium ICPMS, high-resolution
ICPMS, or time-of-flight ICPMS (Montaser, 1998). The ability
to accurately monitor the major isotope of Cr at m/z — 52
should also serve to enhance the sensitivity of this technique by
approximately a factor of 10.
The relative concentration of Cr in the Cr-DNA samples
was determined by using the method of standard additions (1
point addition). Aliquots of the Cr-DNA solutions were diluted
1:1 (v/v) with 2% high purity nitric acid in 18-Mfi-cm distilled
deionized water. Four 20-/xl repeated injections of the diluted
Cr-DNA samples were performed (Fig. 2). Immediately fol-
263
QUANTITATION OF Cr-DNA ADDUCTS BY ICPMS
TABLE 2
Peak-to-Peak Reproducibility for /nFIA-DIHEN-ICPMS
for Several Elemental Isotopes.
Precision
Element
Mass
Natural
abundance"
Peak area
(% RSD)
Peak height
(% RSD)
c
3
o
5
o
4-
g
O
Cr
Cr
As
Pb
Pb
Pb
50
53
75
206
207
208
4.345
9.50
100
24.1
22.1
52.4
2.5
2.8
0.9
1.9
1.6
1.8
2.3
1.9
1.5
0.9
1.8
1.3
en
U
3
-i
it
£H
\
c
.SP
1
GiM)
Note. Precision is expressed as the percent relative standard deviation.
Utilizing a 20-jxl sample loop and 200 pg of element per injection. N = 10.
"From Lide(1995).
lowing the injections of the diluted Cr-DNA sample, an aliquot
of that particular Cr-DNA sample diluted 1:1 (v/v) with a 10
ppb Cr standard in 2% HN0 3 was injected four times, thus
providing a matrix matched 5 ppb standard addition. Peak-topeak reproducibility ranged from 1.0 to 3.2 and 0.4 to 3.8%
RSD (N = 4) based on peak areas and peak heights, respectively. The relative concentration of Cr determined in the
Cr-DNA samples is shown in Table 3. The method detection
limit is approximately 29 fmol of Cr/injection, based on a
20-fjA sample.
Using the concentration of DNA in the solutions, the absolute chromium concentrations were converted to the frequency
of Cr-DNA adducts per 10,000 bases. The dose-dependent
formation and detection of Cr-DNA adducts is shown in Fig.
3. Detection of 0.5 ppb Cr in the untreated control DNA
solution is equivalent to a Cr concentration of 10 nmol/L or
approximately 2 Cr adducts per 10,000 bases. At a Na 2 Cr0 4
concentration of 25 /xM, the frequency of Cr adducts doubled
to 4 adducts per 10,000 bases. This trend increased in a linear,
dose-dependent manner, to a sodium chromate concentration
of 300 JLIM (Fig. 3). The linear dose-response indicates that
Cr(VI) is consumed by the cells and metabolically reduced to
Cr(III) which forms Cr-DNA adducts in a dose-dependent
manner. The frequency of Cr adducts peaks at approximately
20 to 25 Cr-DNA adducts/10,000 bases at 300 and 1000 /xM
Na 2 Cr0 4 , indicating that at these doses HLF are saturated in
their capacity to take up and reduce chromium. This may be
attributed to the depletion of Cr(VI)-reducing moieties such as
ascorbate and glutathione and cell death at the higher chromium concentrations. We have previously reported that
Na2CrO4 concentration levels of 300 and 1000 /xM are mutagenic but result in less than 10% cell survival (Patierno et al.,
1988; Blankenship et al., 1994). Our data suggest that as few
as 20 Cr-DNA adducts per 10,000 bases may contribute to
cellular demise in HLF.
FIG. 2. Peak profiles in juFIA-DIHEN-ICPMS analysis of Cr-DNA extracted from HLF treated with varying concentrations of Na 2 Cr0 4 for 2 hours
(peak profiles corresponding to the Cr-DNA samples with a 5 ppb standard
addition are not shown).
DISCUSSION
In light of our previous reports on the cellular consequences
of Cr-DNA adducts (Xu et al., 1994, 1996), the sensitive
detection of these lesions can act as an important biomarker for
chromium exposure. The doses of sodium chromate used in
this study, 0, 25, 75, 150, 300, and 1000 /xM, produced adduct
levels (background-corrected) of 2.0, 4.0, 7.2, 12.4, 20.3, and
25.2 per 10,000 bases and relative clonogenic survival of 100,
85, 40, 20, 10, and >5%. Based on our published studies, a
sodium chromate dose range of 75 to 300 /aM is similar to the
chromium exposure parameters that are necessary to produce
DNA damage (Xu et al., 1994), inhibition of DNA and RNA
synthesis, cell cycle arrest (Xu et al., 1996), mutations and
neoplastic transformation (Patierno et al, 1988), and apoptosis
(Blankenship et al., 1997) in cultured cells. This relationship is
TABLE 3
Relative Concentration of Cr Determined in Sodium
Chromate-Treated HLF DNA
Na2CrO4
concentration used in
HLF treatment (jxM)
0
25
75
150
300
1000
Cr
concentration
(ppb)
0.51
0.98
1.72
2.91
4.75
5.89
±0.02
± 0.05
±0.11
±0.10
±0.14
±0.19
264
SINGH ET AL.
u
3
•a
•v
a
200
400
600
800
1000
1200
uM sodium chromate
FIG. 3. A plot of the frequency of Cr-DNA adducts as a function of
Na 2 CrO 4 dose. Fitted line is a sigmoidal (Boltzmann) best fit (x2 = 0.05245)
and error bars represent 1 cr.
especially significant because numerous studies have clearly
demonstrated that the mutagenic and transforming activity of
hexavalent chromium can only be observed at doses that also
produce apoptotic cell death and a clear decrease in cell survival. This observation has stimulated much interest in exploring the relationship between chromium-induced apoptosis and
carcinogenesis. Cr-DNA adducts are the precursors for Crinduced DNA-DNA interstrand crosslinks (DDC) detected in
sodium chromate-treated HLF (Xu et ai, 1996). DDC mediate
the guanine-base-specific arrest of several eukaryotic and prokaryotic DNA polymerases (Bridgewater et ai, 1994a,b,
1995). DNA polymerase arrest, in turn, may contribute to the
observed blockage of the cell cycle in the S phase and trigger
apoptotic death (Xu et ai, 1996; Blankenship et ai, 1997).
The detection of Cr-DNA adducts in untreated HLF is
probably related to the fact that Cr is as essential trace element
and, therefore, present in the culture medium. Intracellular
equilibration of Cr is likely to result in a gradual formation of
a low level of Cr-DNA adducts in cells. The fact that our
ICPMS assay is able to detect Cr-DNA adducts in control cells
is a clear indicator of the sensitivity of this technique. Other
factors that may contribute to the observed Cr levels are
isobaric interference of 37C116O and 35C1I8O species and traces
of Cr in the reagents used for extracting DNA.
The present data document that the /u-FIA-DIHEN-ICPMS
technique is capable of detecting the Cr-DNA adducts formed
in cells exposed to a given range of Cr concentration in a
linear, dose-dependent fashion. The data also indicate that this
technique is sensitive to the cellular saturation kinetics of
Cr-DNA adduct formation at high, toxic doses of sodium
chromate. In other words, the maximum frequency of Cr-DNA
adduct formation within the cell is well within the linear
detection range of ICPMS. This feature makes this technique
suitable for measuring Cr-DNA adducts as a biomarker for Cr
exposure. Under similar conditions, the ICPMS technique is
two- to threefold more sensitive in detecting Cr-DNA adducts
in HLF than the radioactive method using 51Cr (Xu et ai,
1996). Furthermore, the ICPMS method has at least threefold
greater linear range than the radioactive method which is
limited by the well-known isotopic dilution problem.
Because Cr often afflicts human beings in concert with other
metals or other environmental and occupational contaminants,
it is difficult to interpret nonspecific risk assessment studies.
For example, Cr and Ni are two of the several toxic components in welding fumes, and both of these metals elicit DPC in
cells (IARC, 1990). Therefore, the small increase in DPC in
workers exposed to welding fumes cannot be definitively attributed to Cr exposure alone (Costa et ai, 1993). In contrast,
the present ICPMS method is potentially a powerful risk assessment tool because it has the capacity of discerning the
exposure to individual metals such as Cr in an environmental
or occupational mixture. Furthermore, this method is rapid, it is
amenable to automation for routine, high throughput human
risk assessment studies, and it does not produce radioactive
waste.
In summary, we have documented that quantitation of CrDNA adducts by the JLAFIA-DIHEN-ICPMS technique offers a
sensitive, direct, nonradioactive method for assessing chromium exposure using less than 1 /ng of DNA. To the best of
our knowledge, this is the first report of using ICPMS for
assessing exposure to Cr by measuring Cr-DNA adducts. This
methodology is not confounded by several variables that are a
handicap for the existing metal biomonitoring techniques. At
the cellular level, this assay system can be conveniently
adapted to measure the rate of repair of metal-DNA adducts
and may allow investigators to explore the mechanisms of
Cr-induced cytotoxicity and cellular transformation. Such studies are currently under way in our laboratory. The applicability
of this technique in cellular studies indicates that it can be used
for animal and human studies of Cr toxicity and carcinogenesis, and for biomonitoring human exposure to chromium. For
example, it is relatively convenient to purify 1 /xg of DNA
from blood lymphocytes of Cr-exposed experimental animals
or people. Importantly, this sensitive technique can be extended to other toxic and carcinogenic metals such as Pb, Cd,
Hg, Cu, and Ni.
ACKNOWLEDGMENTS
The biological and toxicological portion of this research was sponsored by
the National Institutes of Environmental Health Sciences Grant ES-05304 and
by the Elaine H. Snyder Cancer Research Trust to S.R.P. The ICPMS portion
of this research was sponsored by grants from the U.S. Department of Energy
(DE-FGO2-93ER14320) and the National Science Foundation (CHE-9505726
and CHE-9512441) to A.M., and J E Meinhard Associates, Inc., Scholarship
support for J.A.M. was provided by the ARCS foundation. The authors thank
QUANTITAT10N OF Cr-DNA ADDUCTS BY ICPMS
265
Jose Ruiz for his expert technical assistance in the preparation of the Cr-DNA
samples.
Horlick, G., and Montaser, A. (1998). Analytical characteristics of ICPMS. In
Inductively Coupled Plasma Mass Spectrometry (A. Montaser, Ed.). Wiley,
New York.
REFERENCES
IARC (1990). I ARC Monographs on the Evaluation of the Carcinogenic Risk
to Humans: Chromium, Nickel and Welding, Vol. 49. IARC, Lyon, France.
Blankenship, L. J., Carlisle, D. L., Wise Sr., J. P., Orenstein, J. M., Dye, L. E.,
Ill, and Patierno, S. R. (1997). Induction of apoptotic cell death by paniculate lead chromate: Differential effects of vitamins C and E on genotoxicity
and survival. Toxicol. Appl. Pharmacol. 146, 270-280.
Bridgewater, L. C , Manning, F. C. R., Woo, E. S., and Patierno, S. R. (1994a).
DNA polymerase arrest by adducted trivalent chromium. Mol. Carcinog. 9,
122-133.
Bridgewater, L. C , Manning, F. C. R., and Patierno, S. R. (1994b). Basespecific arrest of in vitro DNA replication by carcinogenic chromium:
Relationship to DNA interstrand crosslinking. Carcinogenesis 15, 24212427.
Kuykendall, J. R., Kerger, B. D., Jarvi, E. J., Corbett, G. E., and Paustenbach,
D. J. (1997). Measurement of DNA-protein crosslinks in human leukocytes
following acute ingestion of chromium in drinking water. Carcinogenesis
17, 1971-1977.
Bridgewater, L. C , Manning, F. C. R., and Patierno, S. R. (1995). Chromiummediated DNA interstrand crosslinks cause base-specific DNA polymerase
arrest. Proc. Am. Assoc. Cancer Res. 36, 850.
Bonetti, A., Apostoli, P., Zaninelli, M., Flavia, P., Colombatti, M., Cetto, G. L.,
Franceschi, T., Sperotto, L., and Leone, R. (1996). Inductively coupled
plasma mass spectroscopy quantitation of platinum-DNA adducts in peripheral blood leukocytes of patients receiving cisplatin- or carboplatin-based
chemotherapy. Clin. Cancer Res. 2, 1829-1835.
Bukowski, J. A., Goldstein, M. D., and Johnson, B. B. (1991). Biological
markers in chromium exposure assessment: confounding variables. Arch.
Environ. Health 46, 230-236.
Costa, M., Zhitkovich, A., and Toniolo, P. (1993). DNA-protein cross-links in
welders: Molecular implications. Cancer Res. S3, 460-463.
Costa, M., Zhitkovich, A., Gargas, M., Paustenbach, D., Finley, B., Kuykendall, J., Billings, R., Carlson, T. J., Wetterhahn, K., Xu, J., Patierno, S. R.,
and Bogdanffy, M. (1996). Interlaboratory validation of a new assay for
DNA-protein crosslinks. Mutal. Res. 369, 13-21.
De Flora, S., Camoirano, A., Bagnasco, M., Bennicelli, C , Corbett, G. E., and
Kerger, B. D. (1997). Estimates of the chromium (VI) reducing capacity in
human body compartments as a mechanism for attenuating its potential
toxicity and carcinogenicity. Carcinogenesis 18, 531-537.
Denizeau, F., and Marion, M. (1989). Genotoxic effects of heavy metals in rat
hepatocytes. Cell Biol. Toxicol. 5, 15-25.
Finley, B. L., Kerger, B. D., Katona, M. W., Gargas, M. L., Corbett, G. C , and
Paustenbach, D. J. (1997). Human ingestion of chromium (VI) in drinking
water: Pharmacokinetics following repeated exposure. Toxicol. Appl. Pharmacol. 142, 151-159.
Freeman, N. C. G., and Lioy, P. J. (1997). Exposure to chromium dust from
homes in a chromium surveillance project. Arch. Environ. Health 52,
213-226.
Goyer, R. A., and Cherian, M. G. (Eds.) (1995). Toxicology of metals:
biochemical aspects. In Handbook Experimental Pharmacology, Vol. 115.
Springer-Verlag, New York.
Lide, D. R. (Ed.) (1995). CRC Handbook of Chemistry and Physics. CRC
Press, Boca Raton, FL.
Mattagajasingh, S. N., and Misra, H. P. (1996). Analysis of EDTA-chelatable
proteins from DNA-protein crosslinks induced by the carcinogenic chromium (VI) in cultured intact human cells. Fundam. Appl. Toxicol. 30, 13.
McLean, J. A., Zhang, H., and Montaser, A. (1998). A direct injection high
efficiency nebulizer for inductively coupled plasma mass spectrometry.
Anal. Chem. 70, 1012-1020.
Miller, C. A., Ill, and Costa, M. (1990). Immunodetection of DNA-protein
crosslinks by slot blotting. Mutat. Res. 234, 97-106.
Montaser, A. (Ed.) (1998). Inductively Coupled Plasma Mass Spectrometry.
Wiley, New York.
Montaser, A., Minnich, M. G., McLean, J. A., Liu, H., Caruso, J. A., and
McLeod, C. W. (1998). Sample introduction in ICPMS. In Inductively
Coupled Plasma Mass Spectrometry (A. Montaser, Ed.). Wiley, New York.
Patierno, S. R., Banh, D., and Landolph, J. R. (1988). Transformation of
C3H/10T1/2 mouse embryo cells to focus formation and anchorage independence by insoluble lead chromate but not soluble calcium chromate:
Relationship to mutagenesis and internalization of lead chromate particles.
Cancer Res. 48, 5280-5288.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A
Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY.
Snow, E. T. (1992). Metal carcinogenesis: mechanistic implications. Pharmacol. Ther. 53,31-65.
Xu, J., Bubbley, G. J., Detrick, B., Blankenship, L. J., and Patierno, S. R.
(1996). Chromium(VI) treatment of normal human lung cells results in
guanine-specific DNA polymerase arrests, DNA-DNA crosslinks, and Sphase blockade of the cell cycle. Carcinogenesis 17, 1511-1517.
Xu, J., Manning, F. C. R., and Patierno, S. R. (1994). Preferential formation
and repair of chromium-induced DNA adducts and DNA-protein crosslinks
in nuclear matrix DNA. Carcinogenesis 15, 1443-1450.
Zhitkovich, A., and Costa, M. (1992). A simple, sensitive assay to detect
DNA-protein cross-links in intact cells and in vivo. Carcinogenesis 13,
1485-1489.
Zhitkovich, A. Voitkun, V., and Costa, M. (1995). Glutathione and free amino
acids form stable complexes with DNA following exposure of intact mammalian ceils to chromate. Carcinogenesis 16, 907-913.