The Toxicologist - Society of Toxicology

sion of p-p38, p-p53, p21 and cyclin B1, as well as partially reversing BrO 3-
-induced
G2/M arrest; however, ascorbic acid failed to alter the formation of 8-OHdG
induced by BrO 3-
. On the other hand, N-acetyl-cysteine (NAC), an intracellular
precursor of GSH, enhanced the effect of BrO 3-
on the formation of 8-OHdG.
Surprisingly, NAC partially reversed BrO 3-
-induced G2/M arrest. These data support
the novel finding that ROS mediated MAPK activation is involved in BrO 3-
-
induced cell cycle arrest and that the production of ROS is independent of the formation
of 8-OHdG. It also supports the hypothesis that GSH plays a dual role in
BrO 3-
-induced renal toxicity. This work was supported by Awwa RF 4042, IOA,
MWD, NWRI, Calleagas Water, Long Beach Water, SNWA, LADWP, Veolia,
Environment Abu Dhabi.
812 IN VIVO DISPOSITION AND NEPHROTOXICITY OF
BROMATE IN F344 RATS.
N. Kolisetty 1 , S. Muralidhara 1 , R. J. Bull 2 , O. Quiñones 3 , Z. X. Guo 4 , S. A.
Snyder 3 , J. A. Cottruvo 5 , J. W. Fisher 6 , C. N. Ong 7 and B. S. Cummings 1 .
1
Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA, 2 Mo
Bull Consulting, Richland, WA, 3 Water Quality Research and Development, Southern
Nevada Water Authority, Henderson, NV, 4 Center for Adv. Water Tech., Public
Utilities Board, Singapore, Singapore, 5 Joseph Cotruvo & Assoc., LLC, Washington,
DC, 6 Environment Health Sci., University of Georgia, Athens, GA and 7 Epid. and
Public Health, National University of Singapore, Singapore, Singapore.
The kinetics of bromate absorption and excretion after oral exposures were correlated
to renal and thyroid cell proliferation, death and DNA damage to better understand
the mode of action of this suspected human carcinogen. Exposure of male
and female rats to bromate did not increase bromate blood levels after 28 days compared
to controls. In contrast, exposure of male rats to bromate increased bromate
in the urine from 26 μg/L in controls, to 4,236 and 20,016 μg/L in rats dosed with
125 and 400 mg/L bromate, respectively. Bromide, a bromate degradation product,
increased in the urine of male rats from 58,400 μg/L in controls to 85,522 and
138,590 μg/L after exposure to 125 and 400 mg/L, respectively. Similar results
were seen in female rats for both bromate and bromide. Cell proliferation, as assessed
by bromodeoxyuridine (BrdU) staining, increased 307 and 437% in male rat
kidneys exposed to 125 and 400 mg/L, but only 5 and 19% in female rats kidneys.
Analysis of renal cell morphology using H&E staining showed greater increases in
glomerular and tubular interstitial proliferation in males exposed to bromate than
females, which correlated to alterations in 8-OHdG staining, a marker of DNA
damage. These data suggest that the disposition and excretion of bromate after oral
exposures are similar in male and female rats, which contrasts to its ability to preferentially
induce nephrotoxicity in male rats. This is in contrast to bromate’s ability
to induce similar levels of thyroid toxicity in male and female rats as determined by
H&E and BrdU staining.
813 ANTIOXIDANT DEFENSE IN RENAL PROXIMAL
TUBULAR CELLS FROM NORMAL AND DIABETIC RATS.
L. H. Lash, D. A. Putt, S. R. Terlecky and Q. Zhong. Pharmacology, Wayne State
University School of Medicine, Detroit, MI.
Diabetic nephropathy is characterized by increased oxidative stress and altered mitochondrial
function. We prepared primary cultures of proximal tubular (PT) cells
and mitochondrial fractions from renal cortex from male Sprague-Dawley rats
made diabetic with streptozotocin (STZ; 60 mg/kg ip), to determine characteristics
of antioxidant defense and susceptibility to chemical injury. PT cells from diabetic
rats 30 days after the STZ injection exhibited higher basal and toxicant-stimulated
levels of reactive oxygen species, higher mitochondrial membrane potential, and
greater susceptibility to chemically induced cytotoxicity as compared to those from
age-matched control rats. Both N-acetyl-L-cysteine (NAC) and a cell-permeable
catalase derivative (Cat-SKL) significantly and equally protected PT cells from both
normal and diabetic rats. Despite the higher level of basal oxidant stress, mitochondria
of PT cells from diabetic rats exhibited 6.7-fold higher content of GSH than
those from control rats. We then determined protein expression levels of several key
antioxidant systems in renal mitochondria from diabetic rats at both 30 and 90
days post-STZ injection and in those from age-matched control rats. Protein levels
of the two mitochondrial GSH transporters were slightly higher in diabetes. While
expression of superoxide dismutase 2 (SOD2) was modestly elevated, that of total
thioredoxin 2 (Trx2) was decreased at 30 days and increased at 90 days. Levels of 3-
nitrotyrosine-modified proteins in mitochondria were somewhat higher at both
times in diabetic rats. Assessment of 4-hydroxy-2-nonenal (HNE)-modified proteins
showed primarily 4 bands, which were mostly increased at 30 days but decreased
at 90 days. These results show that although a classical antioxidant and
GSH precursor, NAC, and a catalase derivative both effectively and equally protect
PT cells from both control and diabetic rats from injury, redox processes and mitochondrial
energetics are markedly altered in the renal PT cell due to diabetes and
change as nephropathy develops.
814 FLOW CYTOMETRIC CHARACTERIZATION OF
MITOCHONDRIAL SUBPOPULATIONS IN THE
KIDNEY.
J. E. Saunders, C. Beeson and R. G. Schnellmann. Pharmaceutical and
Biomedical Sciences, Medical University of South Carolina, Charleston, SC.
Mitochondria in the kidney experience periodic bouts of oxidative stress from
metabolic demand and xenobiotic metabolism resulting in persistent damage to
mitochondria. We hypothesize that damaged mitochondria accumulate making the
kidney more susceptible to failure when stressed by disease or xenobiotic exposure.
To assess mitochondrial morphological and functional changes as a result of oxidative
stress, flow cytometric techniques have been developed to quantify features of
individual mitochondria related to size, calcium concentration, mtDNA content,
respiratory capacity and oxidative damage. Mitochondria from rabbit kidneys were
stained with molecular probes for cardiolipin content (nonyl acridine orange,
NAO) and membrane potential (tetramethylrhodamine, TMRM) and they were
analyzed using flow cytometry. Five mitochondrial subpopulations were identified
from side scatter (SSC) gates and the corresponding percentage of mitochondria
and mean fluorescence of the gated populations for NAO and TMRM were statistically
distinct (N = 13). The mean membrane potential and cardiolipin content
ranged about 100-fold across the five subpopulations. Upon titration with uncoupler,
carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), it was
found that the most polarized subpopulations were more resistant to uncoupling
than less polarized populations. Calcium- and iron/calcium-induced swelling of
mitochondria was measured as changes in light scattering at 540 nm and via flow
cytometry. The swelling is evident in cytometry from changes in both SSC and
mean TMRM fluorescence. Interestingly, the more polarized, smaller subpopulations
are also more resistant to calcium/iron-induced swelling as measured from
SSC or loss in membrane potential. These results demonstrate the presence of functionally
distinct populations in the kidney including subpopulations that are resistant
to stressors implicated in pathological states and during aging.
815 ACUTE DOXORUBICIN (DXR)-INDUCED
NEPHROTOXICITY INVOLVES MASSIVE OXIDATIVE
STRESS AND AN ORGANIZED PERTURBATION OF
MITOCHONDRIA-CENTRIC PRO- AND ANTI-
APOPTOTIC GENES.
T. Lahoti and S. D. Ray. Mol. Toxicology Labs, Division of Pharm Sci, AMS College
of Pharmacy & Health Sciences, Brooklyn, NY.
Anticancer agent Doxorubicin has enjoyed popularity for decades because of its
usefulness in the management of various forms of cancers. However, its cardio- &
hepatotoxic potential has put severe constraints on its clinical use. This study explored
DXR’s potential to cause nephrotoxicity in vivo and whether it involves oxidative
stress (OS). Another important goal was to examine whether OS in kidneys
modulate expression of pro- and anti-apoptotic genes in order to maintain cellular
homeostasis. In order to explore all these events, fed male SD rats (~500g) were injected
a single dose of DXR (12mg/Kg, ip) and sacrificed on day-8. Changes in
body weight, serum chemistry were determined; kidney tissues were analyzed for
MDA concentrations, SOD activity, and genomic injury (%DNA frag). The most
important goal was to evaluate the level of expression of APAF-1, Caspase-3, Bad,
Bax, Bcl-2, Bcl-Xl, p53 and MDM-2 genes in order to coherently link extrinsic and
intrinsic pathways of cell death. Data revealed that DXR-exposed animals showed
significant nephrotoxicity (5.6-fold BUN and 2.65 fold creatinine increase), parallel
elevations in lipid peroxidation (1.7 fold) and genomic DNA fragmentation (2.9
fold) with a proportionate reduction in total SOD activity (0.5 fold) suggesting a
massive induction of OS. Western blot analysis data was the most striking, which
disclosed i) increases in the expression of pro-apoptotic APAF-1, Caspase-3, Bax
and Bad proteins; ii) reduction in the expression of anti-apoptotic Bcl-2 and Bcl-Xl
genes; iii) considerable increase in the expression of p53 and suppression in expression
of its regulator MDM-2. Kidney histopathology vividly mirrored changes in
serum chemistry and tissue biochemistry. In summary, this study for the first time
clearly established a close link between mitochondrial perturbations and cell death
regulating genes during DXR-induced nephrotoxicity, and highlighted DXR’s potential
to cause kidney injury in addition to other organs in clinical settings.
SOT 2010 ANNUAL MEETING 173