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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Laboratory of Kidney and Electrolytes Metabolism, National Heart, Lung, and Blood Institute, Department of Health and Human Services, National Institutes of Health, Bethesda, Maryland
Received December 6, 2002; accepted March 26, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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). The patients have progressive renal
failure and are susceptible to the subsequent development of uroepithelial
tumors. Combination of NSAIDs, often including caffeine, and chronic
antidiuresis were implicated in the toxicity.
In an effort to investigate this toxicity, we previously
(Rocha et al., 2001) tested
effects of salicylic acid (SA), acetaminophen (APAP), and caffeine on mouse
renal inner medullary collecting duct (mIMCD3) cells
(Rauchman et al., 1993). We
found that when mIMCD3 cells are subconfluent, 0.5 mM SA or APAP reduces the
number of viable cells by approximately 50%. The drugs have less effect on
confluent cells, which are proliferating more slowly. We speculated that the
much slower turnover of IMCD cells in vivo
(Sheikh-Hamad et al., 2001)
(Zhang et al., 2002) could
explain why clinical toxicity requires very high doses of these drugs over a
very long period of time. Caffeine greatly potentiated the effect of
acetaminophen, pointing to a potential danger of the mixture. Cyclooxygenase
(COX) inhibitors, indomethacin, and NS-398 did not reduce the cell number
except at concentrations greatly in excess of those that inhibit COX.
Therefore, COX inhibition alone was not toxic. APAP arrested most cells in the
late G1 and S phase and produced a mixed form of cell death with
both oncosis (swollen cells and nuclei) and apoptosis. APAP is reported to
inhibit the synthesis of DNA (Hongslo et
al., 1990) and cause chromosomal aberrations
(Brunborg et al., 1995) due to
inhibition of ribonucleotide reductase. Such effects of APAP might account for
renal medullary cell death in vivo and development of uroepithelial tumors
from surviving cells that have chromosomal aberrations.
mIMCD3 cells have important limitations for such studies, however, which
led us to develop for the present studies a more realistic model for studying
toxicity of drugs for renal inner medullary cells. mIMCD3 cells are
immortalized by expression of SV40
(Rauchman et al., 1993). SV40
drives mIMCD3 cells to proliferate rapidly, even when they are confluent,
which might sensitize them to toxicity of NSAIDs and caffeine, especially
since, as noted above, subconfluent cells are more susceptible than are slower
growing confluent cells. In addition, the mIMCD3 cells are routinely cultured
in medium in which osmolality is 300 mOsmol/kg, whereas the osmolality in the
inner medulla varies over a range of 600 to 1700 mOsmol/kg or more
(Bankir, 1996), depending on
the species and diuretic state. mIMCD3 cells are killed by acute increases in
osmolality to 650 mOsmol/kg or above
(Santos et al., 1998;
Michea et al., 2000).
Furthermore, proliferation affects the tolerance of mIMCD3 cells for high
NaCl. When osmolality is increased from 300 to between 500 and 600 mOsmol/kg
by adding NaCl, p53 activity increases
(Dmitrieva et al., 2000). If
p53 is suppressed, fewer cells survive
(Dmitrieva et al., 2000)
because the premature onset of renewed DNA replication is harmful
(Dmitrieva et al., 2001).
Because of these considerations, we have reinvestigated the effects of SA, APAP, and caffeine using passage-1 rat inner medullary collecting duct (p1rIMCD) cells, which tolerate much higher concentrations of NaCl and urea than do mIMCD3 cells and proliferate more slowly, particularly when they are confluent.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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) were
grown on 100-mm Falcon plastic dishes and used between passages 16 and 20.
Cells were fed with 1:1 DMEM-Ham's F-12 medium (Irvine Scientific, Santa Ana,
CA) that contained 2 mM L-glutamine and 10% fetal bovine serum at
37°C in 5% CO2. The osmolality of the basal medium was 300
± 5 mOsmol/kg, as verified using a vapor pressure osmometer (model
5500; Wescor, Logan, UT). The cells were harvested by trypsinization in
Ca2+/Mg2+-free Dulbecco's
phosphate-buffered saline (DPBS) and seeded (12,000 cells/chamber) in
eight-chamber plastic slides (Nalge Nunc International, Naperville, IL). Primary Cultures of p1rIMCD Cells. Pathogen-free male Sprague-Dawley rats (body weight 175 to 250 g; Taconic Farms, Germantown, NY) were killed by decapitation. Their kidneys were immediately excised and processed aseptically as follows. Each kidney was placed in DPBS (pH 7.4; Invitrogen, Carlsbad, CA) at 300 mOsmol/kg, 640 mOsmol/kg (130 mM NaCl and 80 mM urea added), or 1370 mOsmol/kg (225 mM NaCl and 620 mM urea added). The elevated NaCl and urea were maintained throughout processing, tissue culture, and the experimental procedure. The renal inner medulla was quickly dissected and was minced (1–2 mm). The minced tissue was digested for 90 min at 37°C under continuous agitation (300 rpm) in a humidified incubator (5% CO2/95% O2)in50 ml of DMEM/F-12 without phenol red (Invitrogen), containing100 mg of collagenase B (Roche, Indianapolis, IN) and 35 mg of hyaluronidase (Worthington Biochemical, Lakewood, NJ). The resulting cell suspension was centrifuged at 160g for 1 min, washed three times in prewarmed DMEM/F-12 without added enzymes, and resuspended in the tissue culture medium: 50% DMEM low glucose, 50% Coon's Improved F-12 (Mediatech Cellgro, Herndon, VA), 10 mM HEPES, 2 mM L-glutamine (Invitrogen), penicillin G (100 U/ml), streptomycin sulfate (100 µg/ml), 50 nM hydrocortisone, 5 pM 3,3,5-triiodo-L-thyronine, 10 nM sodium selenate, 5 mg/l transferrin, 10% fetal bovine serum (v/v), and added NaCl and urea, as above. The cells from four kidney medullas were plated on a 6 or 10-cm (300 or 640 mOsmol/kg) or 3.5-cm (1370 mOsmol/kg) plastic Petri dish (Corning Incorporated, Corning, NY). Cells were fed each day.
Both subconfluent and confluent cells were studied at 640 mOsmol/kg. The cells were harvested from the dishes by trypsinization in Ca2+ /Mg2+-free DPBS. To study subconfluent cells at 640 mOsmol/kg, 7,000 were seeded in each of eight chambers on a plastic slide (Nalge Nunc International, Naperville, IL) or human fibronectin- or collagen type-I-coated glass, eight-chamber slides (BD Biosciences, San Jose, CA). Then, after 6 h for attachment, experimental media were substituted. To study confluent cells at 640 mOsmol/kg, 90,000 cells were seeded in each chamber. The cells were confluent after 48 h, then experimental media were substituted. Twenty-five thousand cells were seeded in each chamber at 1,370 mOsmol/kg and allowed to attach overnight before substituting the experimental media. At 1,370 mOsmol/kg, proliferation was very slow, and the cells generally did not become confluent. The osmolality of all media was verified with a freezing-point osmometer (Advanced Instruments Inc., Norwood, MA).
Laser Scanning Cytometry (LSC) and Photography. Cells that had been fixed and stained were examined through an epifluorescence microscope with a 20x objective (Olympus BX50; Melville, NY), analyzing them with an LSC (Compucyte Corp., Cambridge, MA) or photographing them with a Kodak Digital Science DC 120 Zoom digital camera (Eastman Kodak, Rochester, NY).
To characterize the cell type, they were fixed with 500 µl of methanol
at –20°C for 2 h, washed 3 times (5 min each) with 500 µl of TBST
(50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) at room temperature,
and incubated for 1 h in 5% bovine serum albumin (fraction V; Sigma-Aldrich,
St. Louis, MO) in TBST. The cells were then incubated with mouse monoclonal
anti-
-smooth muscle actin conjugated with Cy3 (1:100 dilution, clone
1A4; Sigma-Aldrich) and/or mouse monoclonal anti-pan cytokeratin (1:100
dilution, clone C11; Sigma-Aldrich). The nuclei were counterstained with
propidium iodide (0.5 µg/ml; Sigma-Aldrich) in DPBS containing ribonuclease
A (1.0 mg/ml; Sigma-Aldrich). The cells were finally mounted in SlowFade or
ProLong antifade solution (Molecular Probes, Inc., Eugene, OR), photographed,
and analyzed by LSC.
To quantitate the cell number and analyze the cell cycle, cells grown on the eight-chamber permanox slides were fixed in 100% methanol at –20°C for 20 min, permeabilized with 0.1% Triton X-100, incubated with 1 mg/ml ribonuclease A (Sigma-Aldrich) for 15 min, stained with 20 µg/ml propidium iodide (PI) for 5 min, mounted with 150 µl of SlowFade antifade solution (Molecular Probes, Inc.), and analyzed by LSC. The LSC was used to count the number of cells (nuclei) and quantify the PI staining in each nucleus. The integral of PI fluorescence in each nucleus (total PI fluorescence), which is proportional to DNA content, was used to determine position in the cell cycle (G0/G1, S, or G2/M). The data are displayed as cytograms, plotting the number of cells versus the integral of DNA content, and were analyzed using Wincyte software (Compucyte Corp., Cambridge, MA).
Cyclooxygenase Activity. Cells were incubated in control medium or medium containing indomethacin for 2 days. Arachidonic acid (5 µM) (lot 12284h; Cayman, Ann Arbor, MI) was added during the last 24 h. PGE2 in the medium was measured by immunoassay (Cayman), according the manufacture's instructions. Briefly, 50 µlof medium or serial dilutions of PGE2 standards were incubated at room temperature for 18 h in wells coated with PGE2 antiserum and acetyl-cholinesterase-labeled tracer. The reaction mixture was decanted, and the wells were rinsed with wash-buffer, and then 200 µl of Ellman's reagent, containing substrate for the acetylcholinesterase. The enzyme reaction was carried out for 90 min with slow shaking at room temperature. Then, using a Labsystems Multiskan MCC/340 microplate scanning spectrophotometer (Helsinki, Finland), the plates were read at a 415 nm. The PGE2 concentration is expressed in nanograms per milliliter per milligram of cell protein (bicinchoninic acid protein assay; Pierce, Rockford, IL).
Analysis of Mitosis by Immunostaining with Anti-Phospho-Histone H3
Antibody. After fixation in 100% methanol at –20°C for 45 min,
the cells were washed 3 times for 5 min each with 0.1% Triton X-100 in PBS,
followed by blocking buffer (3% bovine serum albumin-0.1% Triton X-100). Then
they were incubated with anti-phospho-histone H3 (mitosis marker) (no. 06-570;
Upstate Biotechnology, Lake Placid, NY) antibody, washed with 0.1% Triton
X-100 in PBS, incubated for 1 h with secondary antibody (Alexa 488
goat-anti-rabbit IgG, no. A-11034; Molecular Probes), stained with 0.7
µg/ml PI and mounted with 150 µl of antifade (S-7461; Molecular Probes).
The slides were analyzed by LSC. Green fluorescence integral was recorded to
measure anti-phospho-histone H3 antibody binding (P-histone H3 content). Red
fluorescence was recorded to measure PI binding (DNA content). Bivariate
distributions of cells showing P-histone H3 content versus DNA content were
obtained, and the percentage of P-histone H3 positive (mitotic) cells was
determined (Juan et al.,
1998).
Analysis of Proliferating Cell Nuclear Antigen (PCNA) Abundance and of Histone H2AX Phosphorylation by Immunostaining. The immunostaining procedure was the same as for anti-phospho-histone H3, except that the primary antibodies were anti-phospho-H2AX(Ser139) (no. 07-164; Upstate Biotechnology) or anti-PCNA (PC10) (Santa Cruz Biotechnology, Santa Cruz, CA). Using LSC, the cells were analyzed for green fluorescence to measure anti-PCNA or anti-phospho-histone H2AX antibody binding and red fluorescence to measure PI binding (DNA content). Bivariate distributions of cells showing maximal intensity of green fluorescence in a nucleus (Green Max Pixel) versus DNA content were obtained. The limits of the region of the cytogram containing cells with high of Green Max Pixel (PCNA or phospho-H2AX positive cells) was determined by eye based on the control sample and the percentage of cells in that region (PCNA or phospho-H2AX-positive cells) was calculated.
Hoechst Staining. p1rIMCD cells were plated in an eight-chamber slide and allowed to grow for 2 days to reach confluence and then treated with 2 mM of the indicated NSAIDs for 18 or 24 h. The cells were fixed in 10% formalin (Fisher Scientific, Fair Lawn, NJ) for 15 min and washed 3 times with PBS followed by staining with 10 µg/ml Hoechst-33258 DNA dye (Molecular Probes). After staining, the slides were mounted with antifade solution (Molecular Probes).
Drugs. APAP, caffeine, SA, and indomethacin ere from Sigma-Aldrich. All drugs were directly dissolved in medium except indomethacin, which was dissolved in dimethyl sulfoxide (DMSO) and then diluted with medium to a final DMSO concentration of 0.1% or less. The same concentration of DMSO was added to control cells, as appropriate.
Statistical Methods. In each individual experiment, each condition was analyzed in triplicate (three separate chambers on slides). Then the results from the individual experiments were averaged and are presented as the mean ± S.E.M. (n = number of individual experiments). Significance was analyzed with the GraphPad Instat program (GraphPad Software, San Diego, CA), using analysis of variance completed by Dunnett's multiple comparisons post-test, according to the number of experiments. A p value <0.05 is considered significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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), which identifies them
as IMCD cells.
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LSC was used for cell cycle analysis of subconfluent and confluent mIMCD3 in 300 mOsmol/kg medium and of p1rIMCD cells prepared and grown in media at 300, 640, or 1370 mOsmol/kg (Table 1). Proliferating cells are in the S or G2/M phases of the cell cycle. Approximately 34% of confluent mIMCD3 cells are in S and G2/M, consistent with continued proliferation that persists even after the cells become confluent. In contrast, only approximately 14 to 25%, depending on the osmolality, of confluent rat primary inner medullary cells are in S and G2/M, consistent with more effective contact inhibition of proliferation. Also, the percentage of confluent cells in S, i.e., those that are replicating DNA, is much less for p1rIMCD (2 to 6%) than for mIMCD3 cells (20%). Furthermore, confluent mIMCD3 cells tend to pile up in multiple layers after they are confluent, whereas the primary rat inner medullary cells do not (not shown). We conclude that the percentage of confluent cells that are in the process of replicating DNA and proliferating is much smaller for p1rIMCD cells than for mIMCD3 cells.
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Effect of APAP, SA, and Caffeine on Subconfluent p1rIMCD Cells at 640
mOsmol/kg. When p1rIMCD cells are prepared and grown at 640 mOsmol/kg, 4
days of SA, APAP, or caffeine significantly reduces the number of subconfluent
p1rIMCD cells (Fig. 2A). A
higher concentration of SA or APAP is required to reduce the number of
subconfluent p1rIMCD cells than previously observed for subconfluent mIMCD3
cells (Rocha et al., 2001). SA
or APAP (1.0 to 2.0 mM) reduces the subconfluent p1rIMCD cell number by
approximately 50%, whereas only 0.5 mM is required to reduce the mIMCD3 cell
number to the same extent. On the other hand, less caffeine is necessary to
reduce the subconfluent p1rIMCD cell number than to reduce the mIMCD3 cell
number. More than 1.0 mM caffeine is required to reduce the subconfluent
mIMCD3 cell number significantly, whereas 0.5 mM caffeine reduces the
subconfluent p1rIMCD cell number significantly.
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The effects of APAP and caffeine on subconfluent p1rIMCD cells are
additive. APAP (0.5 mM) plus caffeine (0.5 mM) reduces the p1rIMCD cell number
significantly more than either drug alone
(Fig. 2B). In contrast, adding
SA to APAP and/or caffeine does not further reduce the cell number. The
previous results with subconfluent mIMCD3 cells were qualitatively similar
(Rocha et al., 2001). As
little as 0.1 mM caffeine adds significantly to the effect of 0.5 mM SA plus
0.5 mM APAP (Fig. 2C).
Reduction of the cell number was associated with a decrease in the percentage
of mitotic cells (Fig. 2D) and
an increase in the percentage of cells with damaged DNA (P-H2AX staining;
Fig. 2E). The reduction in the
cell number was evident after 1 day (Fig.
2F).
Effect of APAP, SA, and Caffeine on Confluent p1rIMCD Cells at 640
mOsmol/kg. When the p1rIMCD cells are confluent at 640 mOsmol/kg, 4 days
of 2.0 mM SA or caffeine does not affect the cell number significantly
(Fig. 3A), similar to the lack
of effect of SA and caffeine, singly or combined, on confluent mIMCD3 cells
(Rocha et al., 2001).
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When p1rIMCD cells are confluent at 640 mOsmol/kg, APAP has a strikingly
different effect (Fig. 3) from
that on confluent mIMCD3 cells (Rocha et
al., 2001) and that on subconfluent p1rIMCD cells
(Fig. 2A). Whereas 2.0 mM APAP
reduces the number of subconfluent p1rIMCD cells and does not significantly
affect number of confluent mIMCD3 cells, it significantly increases the number
of confluent p1rIMCD cells at 640 mOsmol/kg
(Fig. 3A). The increase is even
larger when both APAP and caffeine are added
(Fig. 3A).
DNA replication and cellular proliferation generally are accompanied by
expression of PCNA (Fairman,
1990; Iatropoulos and
Williams, 1996). APAP increases the percentage of cells expressing
PCNA (Fig. 3B), consistent with
increased proliferation. DNA damage is generally is accompanied by increased
phosphorylation of histone H2AX (Redon et
al., 2002). Associated with increased proliferation of p1rIMDC
cells, APAP increases the percentage of cells expressing P-H2AX
(Fig. 3C), indicative of DNA
damage. Nevertheless, there is no apparent change the appearance of the nuclei
stained with the Hoechst reagent (Fig.
3D).
When p1rIMCD cells are confluent at 640 mOsmol/kg, 2.0 mM SA plus 2.0 mM caffeine or a combination of APAP, SA,and caffeine at 2.0 mM each significantly reduces the cell number (Fig. 3A), which is accompanied by nuclear condensation and fragmentation (Fig. 3D), indicative of apoptosis.
Effects of APAP, SA, and Caffeine on p1rIMCD Cells at 1370 mOsmol/kg. The osmolality in rat inner medullas is approximately 600 mOsmol/kg during water diuresis. During antidiuresis, salt and urea concentrations increase along a gradient of osmolality that rises from approximately 600 mOsmol/kg at the base of the inner medulla to 1700 mOsmol/kg or more at the tip, depending on the duration and severity of dehydration. Therefore, it was of interest to test the effects of the drugs on p1rIMCD cells prepared and grown in media whose osmolality was elevated to 1370 mOsmol/kg by adding NaCl and urea, which is the highest osmolality at which it was practical to grow sufficient cells for the experiments. Proliferation generally was so slow at 1370 mOsmol/kg that the cells did not became completely confluent during the 4 days of the experiment. Under those conditions, APAP (≥0.5 mM) significantly increases the cell number, as do combinations of drugs that include APAP at ≥0.1 mM or more of each drug (Fig. 4A). The increase in the cell number is evident after 2 days (Fig. 4B). It is accompanied by elevated expression of PCNA (Fig. 4C), consistent with increased cellular proliferation, and elevated P-H2AX (Fig. 4D), consistent with increased DNA damage. When both APAP and SA are added, the increase in the cell number (Fig. 4A) is accompanied by nuclear condensation and fragmentation (Fig. 4E), consistent with apoptosis.
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In contrast to the increase in the cell number with APAP and mixtures of drugs containing APAP, ≥1.0 mM caffeine or ≥1.0 mM each of caffeine and SA significantly decreases the cell number and is accompanied by the appearance of cells with condensed and fragmented nuclei (Fig. 4F), characteristic of apoptosis.
The effect of indomethacin was tested to determine how inhibition of COX
might affect the p1rIMCD cells. COX activity in subconfluent p1rIMCD cells at
640 mOsmol/kg was estimated from the appearance of PGE2 in the
medium (Fig. 5). Indomethacin
(10 µM) almost completely inhibits COX activity without any effect on the
cell number, which excludes the possibility that the effects of the other
drugs under these conditions involve COX inhibition. Similar lack of effect of
COX inhibition, per se, was previously observed with mIMCD3 cells
(Rocha et al., 2001).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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) in that
p1rIMCD could be readily tested under conditions of high NaCl and urea like
those normally found in inner medullas. Similar to the effects of the drugs on
subconfluent mIMCD3 cells at 300 mOsmol/kg
(Rocha et al., 2001), SA,
APAP, and/or caffeine reduce the number of p1rIMCD cells when they are
subconfluent at 640 mOsmol/kg (Fig. 2, A
and B). The concentration of SA or APAP needed to reduce the
p1rIMCD cell number (Fig. 2A)
is approximately twice that needed to produce a comparable decrease in the
mIMCD3 cell number (Rocha et al.,
2001), but the concentration of caffeine is approximately half as
great. With both kinds of cells, the drug effects are additive, particularly
the combination of APAP and caffeine (Fig.
2) (Rocha et al.,
2001). It is particularly striking that as little as 0.1 mM
caffeine significantly increases the effect of APAP plus SA to reduce the
p1rIMCD cell number (Fig. 2),
which supports the possibility that the caffeine, taken in excess, could add
to the renal medullary toxicity of the combination of APAP plus SA.
SA and caffeine have a greater effect on mIMCD3 cells when they are
subconfluent at 300 mOsmol/kg than when they are confluent
(Rocha et al., 2001). The same
trend is seen with p1rIMCD cells at 640 mOsmol/kg. SA significantly reduces
the number p1rIMCD cells when they are subconfluent
(Fig. 2, A and B) but not when
they are confluent (Fig. 3A).
Nevertheless, 2.0 mM SA plus 2.0 mM caffeine or 2.0 mM of all three drugs
combined reduces the number of confluent p1rIMCD cells
(Fig. 3A). This is consistent
with our previous conclusion that these agents are more toxic to proliferating
cells than to quiescent ones and that the drugs might be more toxic in
combination than singly. Few cells normally are proliferating in rat inner
medullas (Sheikh-Hamad et al.,
2001; Zhang et al.,
2002). Targeting of toxicity to those few cells is consistent with
the slow onset and progression of analgesic-associated renal disease
([No Authors Listed],
1984).
An unexpected novel result of the present studies is that APAP, alone and in combination with the other drugs, strikingly increases the number of p1rIMCD cells when they are confluent at 640 mOsmol/kg (Fig. 3) or growing very slowly at 1370 mOsmol/kg (Fig. 4) in striking contrast to reduction of the number of p1rIMCD cells that are subconfluent at 640 mOsmol/kg (Fig. 2). Caffeine alone does not affect the number of confluent cells at 640 mOsmol/kg, but it adds greatly to the increase in the cell number caused by APAP (Fig. 3A). The effect of APAP to increase the cell number is even more striking at 1370 mOsmol/kg. APAP (0.5 mM or more), alone, or 0.1 mM or more of APAP combined with 0.1 mM or more of SA and/or caffeine greatly increases the number of cells that otherwise are proliferating very slowly at 1370 mOsmol/kg (Fig. 4A). As discussed above, there is little proliferation of renal inner medullary cells in vivo, and proliferating inner medullary cells have reduced tolerance for hyperosmolality. APAP-induced proliferation appears to sensitize the cells to toxicity, as evidenced by appearance of DNA damage (increased P-H2AX expression; Figs. 3C and 4D) and apoptosis when APAP is added in combination with SA (Fig. 4E).
APAP was previously reported either to increase or decrease proliferation
of various cells. The reports of increased proliferation include that 0.1 to
0.3 mM APAP produces significant growth stimulation of several human tumor
cell lines and one of two normal fibroblast cell lines
(Schonberg and Skorpen, 1997).
It causes a brief burst of mitosis in a glioma cell line, which transiently
increases the cell number, followed by apoptosis which reduces the cell number
(Casper et al., 2000). It also
stimulates proliferation (3H-dT incorporation) of breast cancer cells
(Harnagea-Theophilus et al.,
1999) and increases bromodeoxyuridine incorporation in livers of
hamsters but not of rats (Hiruma et al.,
2001). Treating mice with increasing doses of APAP for 8 days
increases hepatocellular proliferation 4-fold
(Shayiq et al., 1999). Reports
of decreased proliferation include that APAP produces cell cycle block in Hepa
1-6 cells (Boulares et al.,
1999) and HL-60 cells (Wiger
et al., 1997), reduces replicative DNA synthesis in mouse mammary
tumor cells (Hongslo et al.,
1990), and reduces proliferation of HepG2 cells
(Dai and Cederbaum, 1995) It
has not been clear what factors determine whether APAP will increase or
decrease the cell number, but the rate of proliferation and DNA replication
apparently is important in p1rIMCD cells. It would be of interest to know
whether high levels of APAP increase the otherwise slow rate of proliferation
of renal inner medullary cells in vivo.
Mechanisms of the Effects of APAP and Caffeine. NSAIDs inhibit
proliferation of some tumorigenic cells and kill them
(Baron and Sandler, 2000),
which has led to numerous studies of the mechanisms involved. Both
COX-dependent and independent mechanisms have been identified
(Chan et al., 1998;
Baron and Sandler, 2000). Since
we did not find COX inhibition per se to be toxic
(Fig. 5), we will concentrate
on COX-independent mechanisms.
Acute overdoses of APAP cause acute and sometimes fatal liver damage
(Perry and Shannon, 1998). The
toxicity is caused by a minor metabolic product, N-acetyl-p
benzoquinone imine (NAPQI) that attaches to the hepatic cell membranes and
injures the lipid bilayer if not neutralized by an antioxidant. Hepatic
glutathione appears to be the primary antioxidant that conjugates and
neutralizes NAPQI. The resulting oxidative stress in the cell may ultimately
lead to its demise. NAPQI also binds to cell macromolecules, which can cause
cell death (Mirochnitchenko et al.,
1999). However, APAP, has an additional toxic effect that may be
more pertinent to the toxicity that we observe. The drug directly inhibits
ribonucleotide reductase, which reduces cell growth by stopping DNA
replication (Hongslo et al.,
1990). Then, the relative number of cells in S phase increases
(Hongslo et al., 1990), as we
observed following exposure of mIMCD3 cells to APAP
(Rocha et al., 2001). In the
process DNA is damaged, leading to sister chromatid exchange and chromosomal
aberrations. APAP also inhibits nucleotide excision repair
(Brunborg et al., 1995).
Histone H2AX is phosphorylated on Serine 139 in response to double-strand
breaks (Rogakou et al., 1998),
which is one of the first steps in DNA repair
(Paull et al., 2000). That
APAP induces this phosphorylation (Figs.
3C and
4D) supports the conclusion
that, under these conditions, the drug induces DNA double-strand breaks,
causing toxicity.
Caffeine has long been known to have numerous actions
(Serafin, 1996), including 1)
inhibition of phosphodiesterases, thereby increasing intracellular cyclic AMP,
2) direct effects on intracellular calcium concentration, 3) indirect effects
on intracellular calcium concentrations via membrane hyperpolarization, and 4)
antagonism of adenosine receptors. In addition, it has recently become
apparent that caffeine also influences multiple pathways involved in the
cellular response to DNA damage. It reduces DNA damage-induced cell cycle
arrest in G1, S, and G2/M, abolishing the
G2/M checkpoint by inhibiting ATM kinase activity
(Zhou et al., 2000). Caffeine
also blocks p53 activation in response to DNA damage and blocks the repair of
DNA damage (Murnane, 1995).
The result is that caffeine potentiates the lethal effects of ionizing
radiation. We now observe that caffeine strongly potentiates the toxicity of
APAP. The mechanism could be related to the cumulative effects of the two
drugs on DNA damage and repair. The effects of caffeine start at
concentrations close to those that inhibit ATM/ATR kinases
(Sarkaria et al., 1999). These
kinases are activated by DNA damage and coordinate a DNA damage response,
involving accurate DNA repair accompanied by cell cycle arrest
(Zhou and Elledge, 2000).
Thus, inhibition of ATM/ATR kinases by caffeine is a mechanism that might
impair repair of the DNA damage caused by APAP, increasing its toxicity.
Why might these drugs be particularly toxic to renal inner medullary cells?
One factor is that drugs can be concentrated in the inner medulla as a result
of the urinary concentrating mechanism. Thus, during antidiuresis, APAP is 4
times higher in the renal medulla than in peripheral plasma
(Duggin, 1980). We do not know
whether this is also true of caffeine, being unaware of any measurements of
caffeine concentration in renal medullas. A second factor is that the high
osmolality present in renal medullas could sensitize the cells to the drugs.
We previously showed that proliferation sensitizes renal inner medullary cells
to osmotic stress (Zhang et al.,
2002), and the cells die mostly in the S phase of cell cycle,
during DNA replication (Dmitrieva et al.,
2001). High levels of NaCl, like those that occur in the renal
inner medulla, damage DNA (Kultz and
Chakravarty, 2001), particularly during DNA replication, and
inhibit repair of this damage (Dmitrieva
et al., 2003), which possibly adds to DNA damage caused directly
by APAP. In addition, at high osmolality, APAP increases the number of PCNA
positive cells, evidence that it causes the cells to enter S phase under these
conditions, which could sensitize them to the osmotic stress.
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Footnotes |
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ABBREVIATIONS: NSAID, nonsteroidal anti-inflammatory drug; SA, salicylic acid; APAP, acetaminophen; mIMCD3, mouse inner medullary collecting duct 3; COX, cyclooxygenase; NS-398, N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methanesulfonamide; p1rIMCD, passage-1 rat inner medullary collecting duct; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's phosphate-buffered saline; LSC, laser scanning cytometer; PI, propidium iodide; PGE, prostaglandin E; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; DMSO, dimethyl sulfoxide.
1 Present address: Laboratory of Cellular and Molecular Physiology, Faculty
of Medicine, Universidad de los Andes, San Carlos Apoquindo 2200, Santiago,
Chile. 
2 Present address: 8 Tennyson St., Edison, NJ 08820.
3 Present address: Department of Pharmacology, University of Cambridge,
Tennis Court Rd., Cambridge, CB2 1QJ, UK.
Address correspondence to: Maurice B. Burg, NHLBI, NIH, Bethesda, MD 20892-1603. E-mail: maurice_burg{at}nih.gov
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