3. Research back ground
Bromate (BrO3-) is a water disinfection by-product (DBP) and is possibly carcinogenic to humans.
One of the major target organs of BrO3- is the kidney.
Chronic exposure to high doses of BrO3- induces renal adenoma and carcinoma and non-cancerous urothelial hyperplasia. BrO3-
induced renal toxicity has been associated with DNA damage, primarily8-hydroxyl de oxy- guanosine (8-OHdG) formation.
Chlorite is a regulated water disinfection by-product found in g/l concentrations in some disinfected waters.
BCAA is currently an unregulated halo acid, which constitutes one of the most prevalent by-products in drinking water.
BCAA increased incidences of adenomas of the large intestine and in rats, and induced hepatocellular neoplasms and hepatoblastoma.
Non-neoplastic liver lesions were observed in BCAA exposure.
A gene array study suggested that carcinogenic pathways involved in BCAA induced mesothelioma include insulin-like growth factor 1
(IGF-1), p38 MAPK, Wnt/beta-catenin and integrin signaling pathways.
4. Research objectives
To investigate the effect of NaClO2 and BCAA on BrO3--induced DNA damage
and renal cell death.
To increase the understanding of the mode of these chemicals and increase the
knowledge on the mixture on renal toxicity.
5. Reagents and
antidotes
Cell culture
Assessment of
morphology
Assessment of
cell viability
Nuclear
morphology
Measurement of cell
death
Measurement of
cell cycles
Immunoblot
analysis
Immunocyto
chemistry
ROS
measurement
Measurement of
ATP levels
Protein Determination
Statistical
analysis
Experimental design
6. Results
Effect of NaClO2 and BCAA on BrO3--induced alterations in MTT
staining and cell morphology.
This study determined the time- and concentration-dependence
of BrO3- cytotoxicity, and showed that concentrations of BrO3- from
100 to 400 ppm induce necrosis in NRK cells after 48 h of
exposure. BrO3- (200 ppm) moderately decreased MTT staining in
NRK cells after 48 h.
Treatment of NRK cells with NaClO2 alone decreased MTT staining
only at concentrations over 20 ppm. In contrast, treatment of NRK
cells with 10 or 20 ppm NaClO2 simultaneously with BrO3
decreased MTT staining compared to cells exposed to BrO3 alone.
Exposure of NRK cells to only BCAA decreased MTT staining at
concentrations of 50 ppm and above. Treatment of cells with 50 ppm
BCAA simultaneously with BrO3- decreased MTT staining
compared to cells exposed to BrO3- alone.
Exposure of cells to 10 ppm NaClO2 and 50 ppm BCAA
simultaneously with BrO3 significantly decreased MTT staining
compared to cells exposed BrO3- alone.
In contrast, exposure of cells to a mixture
of just NaClO2 and BCAA together resulted in slight decreases in
MTT staining. The effects of NaClO2 and BCAA on BrO3—induced
decreases in MTT staining were confirmed by cell morphology
Effect of NaClO2 and BCAA on BrO3--induced 8-OHdG formation. NRK cells were treated with 200 ppm KBrO3 alone, or in
combination with NaClO2, BCAA or both for 24 h prior to analysis of nuclear morphology and 8-OHdG staining using fluorescence
microscopy. Representative staining for DAPI (shown in blue) and 8-OHdG (shown in green) is depicted in (A) and the percentage
of nuclei staining positive for 8-OHdG after exposure to 100 or 200 ppm BrO3- are shown in (B and C). White arrows in (A) ndicate
nuclei staining positive for 8-OHdG. Data are represented as the mean ± SEM of at least 3 separate experiments. Means with
different subscripts are significantly (P < 0.05) different from each other.
7. Results
Effect of NaClO2 and BCAA on BrO3--induced 8-OHdG
staining.
Treatment of NRK cells with 100 or 200 ppm BrO3-
alone induced moderate increases in 8 OHdG staining
as assessed by fluorescence microscopy.
Exposure of cells to all three DBPs increased 8-OHdG
staining to levels comparable to cells exposed to both
BrO3- and NaClO2.
Effect of NaClO2 and BCAA on BrO3--induced 8-OHdG formation. NRK cells were treated with 200 ppm KBrO3 alone, or in
combination with NaClO2, BCAA or both for 24 h prior to analysis of nuclear morphology and 8-OHdG staining using fluorescence
microscopy. Representative staining for DAPI (shown in blue) and 8-OHdG (shown in green) is depicted in (A) and the percentage of
nuclei staining positive for 8-OHdG after exposure to 100 or 200 ppm BrO3- are shown in (B and C). White arrows in (A)
indicate nuclei staining positive for 8-OHdG. Data are represented as the mean ± SEM of at least 3 separate experiments.
8. Results
Effect of NaClO2 and BCAA on BrO3--induced
alterations in cell cycle.
Exposure to NaClO2 and BCAA alters the cell cycle
arrest induced by BrO3-. Treatment of cells with NaClO2
(10–40 ppm) alone induced a moderate G2/M arrest.
BCAA exposure only slightly altered NRK cell
cycle at concentrations as high as 50 ppm.
Exposure of cells to NaClO2 and BrO3- significantly
decreased the percentage of cells in the G2/M phase of
the cell cycle, compared to cells exposed to
BrO3- alone.
BCAA had no effect on BrO3 induced changes in cell
cycle .
Exposure of cells to a mixture of all three chemicals
yielded results similar to that seen
in cells exposed to BrO3- and NaClO2.
Effect of NaClO2 and BCAA on BrO3--induced cell cycle arrest. NRK cells were treated with 10, 20 and 40 ppm
NaClO2 alone (A) or 20 and 50 ppm BCAA alone (B)for 24 h prior to analysis of cell cycle using PI staining and flow
cytometry. Cells were exposed to 200 ppm KBrO3 alone, or in combination with 20 ppm NaClO2 (C), 20 ppmBCAA
(D), or both (E) for 24 h, prior to analysis of cell cycle. Data are represented as the mean ± SEM of at least 3
separate experiments
9. Effect of NaClO2 and BCAA on BrO3--induced cell death.
we exposed NRK cells to these chemicals alone or in mixtures, and assessed alterations in annexin V
(apoptotic cell marker) and PI (necrotic cell marker) staining using flow cytometry.
Exposure of cells to BrO3- alone increased the percentage of cells staining positive for PI alone, without
increasing the number of cells staining positive for annexin V alone, or for both annexin V and PI (late
apoptosis).
Interestingly exposure to both BrO3- and NaClO2 decreased the percentage of cells staining positive for PI
alone, and increased the percentage of cell staining positive for both annexin V and PI.
Interestingly exposure to both BrO3- and NaClO2 decreased the percentage of cells staining positive for PI
alone, and increased the percentage of annexin V positive cells.
Exposure of cells to BCAA creased the percentage of cells staining positive for both annexin V and PI
ercentage of annexin V positive cells. and BrO3- also decreased the percentage of cells staining positive for
PI alone and increased the number of annexin V positive cells; however, BCAA also in BCAA also
increased the percentage of cells.
Treatment of cells with all three chemicals significantly increased the percentage of cells staining positive
for annexin V alone, and those staining positive for both annexin V and PI. These increases were greater
than those observed in cellsexposed to BrO3- and NaClO2, or BrO3- and BCA
Results
10. Treatment of cells with all three chemicals
significantly increased the percentage of cells
staining positive for annexin V alone, and those
staining positive for both annexin V and PI. These
increases were greater than those observed in cells
exposed to BrO3- and NaClO2, or BrO3- and
BCAA.
we assess DAPI staining and annexin and PI
staining using fluorescence microscopy.
Exposure of cells to BrO3-, NaClO2 and BCAA
resulted in an increase in cells staining positive
for annexin V, with the characteristic halo-like
pattern of apoptosis
Results
Effect of NaClO2 and BCAA on BrO3--induced cell death and nuclear morphology. NRK cells were exposed to 200 ppm KBrO3
alone or in combination with 10 ppm NaClO2 (A), 20 ppm BCAA (B) or both (C) for 48 h prior to analysis of annexin V and PI
staining as determined using flow cytometry. In addition, nuclear morphology (D, upper panel) and annexin and PI staining (D,
lower panel) were assessed using fluorescence microscopy. Cisplatin was used in (D) as a positive control. Data in (A–C) are
represented as the mean ± SEM of at least 3 separate experiments. The arrows represent apoptotic nuclear morphology (D, upper
panel) and the characteristic halo-like staining of annexin V-FITC of the plasma membrane (D, lower panel). Means with different
subscripts are significantly (P < 0.05) different from each other. Data in (D) are representative of at least 3 individual experiments
11. ClO2 and BCAA on BrO3--induced ROS
formation.
Exposure of cells to BrO3 alone increased CM-
H2DCFDA staining compared to control cells.
Exposure of cells to NaClO2 and BCAA alone
also increased CM-H2DCFDA staining.
Exposure of cells to BrO3 and NaClO2, or BrO3-
and BCAA, or a mixture of all three, did not
significantly increase CM-H2DCFDA staining
compared to cells exposed to BrO3- alone.
It suggest that the ability of mixtures of DBPs to
increase cell death is not a result in an increase
in the overall levels of ROS.
Results
Effect of NaClO2 and BCAA on BrO3--induced ROS formation. NRK cells were preloaded with 10 M CM-H2DCFDA for 30 min and then
exposed to NaClO2 (0–50 ppm) (A), BCAA (0–150 ppm) (B) or 200 ppm KBrO3 alone, or in combination with 10 ppm NaClO2 alone, 20
ppm BCAA alone or both (C) for 30 min prior to measurement of fluorescence intensity. Data are represented as the mean ± SEM of at
least 3 separate experiments. Means with different subscripts are significantly (P < 0.05) different from each other.
12. Effect of NaClO2 and BCAA on BrO3--
induced expression of DNA damage and
stress response proteins
Exposure of cells to BrO3- alone increased the
expression of numerous stress response and DNA
damage response proteins, including p38 MAPK
and p21, as well as H2AX phosphorylation
and p53 phosphorylation.
Exposure of cells to all three chemicals increased
the expression of p-p38 compared to BrO3-
alone.
Interestingly, p-p53 expression was similar to
that seen in cells exposed to BrO3- alone, while
the expression of p21 and p-H2AX were
essentially unchanged.
Results
Effect of NaClO2 and BCAA on BrO3--induced alterations in cell signaling protein expression. NRK cells were exposed to 200 ppm
KBrO3 alone, or in combination with 10 ppm NaClO2, 20 ppm BCAA or both for 24 h prior to analysis of protein expression using
immunoblot analysis. p38 is shown as a loading control. All blots are representative of at least 3 separate experiment
13. Effect of NaClO2 and BCAA on BrO3 induced alterations
in ATP.
Treatment of cells with BrO3- alone induced
concentration dependent decreases in ATP levels.
BrO3- concentrations of 200 ppm decreased ATP
levels 20%, which is consistent with the level of
cell death induced at this concentration.
Treatment of cells with 10 ppm NaClO2 decreased
cellular ATP levels 20%, while treatment with 20
ppm BCAA had no significant affect, compared to
control cells.
In contrast, treatment of cells with mixtures
of all three chemicals decreased cellular ATP levels
60% compared to control cells.
Decrease in cellular ATP levels in the presence of
all three chemicals is consistent with decreases in
cell viability .
Results
Effect of NaClO2 and BCAA on BrO3--induced ATP depletion. NRK cells were exposed to 0–400 ppm KBrO3 (A) alone
for 48 h, or to 200 ppm KBrO3 in combination with 10 ppm NaClO2 or 200 ppm BCAA or both (B) for 48 h prior to
analysis of cellular ATP levels using luminescence assays. Data are represented as the mean ± SEM of at least 3
separate experiments
14. Proposed interactions between DBPs and renal cell
death. Exposure of renal cells to BrO3- alone
activates DNA damage-independent and DNA
damage dependent pathways. The DNA-independent
pathway involves a ROS-mediated MAPK pathway
in which p38 activates p53 and p21.
The DNA damage-dependent pathway results in 8-
OHdG formation, which also leads to p53 and p21
activation.
Exposure of cells to BrO3- in the presence of
NaClO2 increases 8-OHdG formation,
p53 activation and p21 expression, as well as H2AX
phosphorylation.
In contrast, exposure of cells to BrO3- in the
presence of BCAA increases p38 and p53 activation
and p21 expression, without increasing 8-OHdG
formation.
The interactions between these DBPs appear to
switch the mechanisms of BrO3--induced renal cell
death from necrosis to apoptosis.
Results
15. conclusion
DBPs synergistically increases cell death
Increases in cell death correlated to increased DNA damage, apoptosis,
alteration in the cell cycle as well as alterations in expression of cells
stress and DNA damage response proteins.
