The Toxicologist - Society of Toxicology

and renal inflammation induced by 20 mg/kg cisplatin, ip, significantly increased
levels of uFg (~4-fold) and Kim-1 (~15-fold) were detected as early as 24 h that
peaked at 72 h (~20-fold for both). In contrast, a rat model of gentamicin (50, 100,
200, or 300 mg/kg, sc, for 3 days) induced proximal tubular damage in the absence
of vascular dysfunction and inflammation, resulted in significant increases in urinary
Kim-1 and NAG, but did not result in any change in kidney Fg mRNA or urinary
Fg. Furthermore, in a human cross-sectional study, a ~2000-fold increase in
uFg was observed in 10 patients with clinically established multifactorial AKI versus
healthy volunteers (n=10) compared to a 20-fold excretion of urinary Kim-1 and
NAG. In a longitudinal follow-up of 2 patients, who underwent surgical repair of
abdominal aortic aneurysm and developed AKI after surgery, increased levels of uFg
were detected earlier than changes in serum creatinine, which supports the potential
use of uFg as predictive biomarker of renal vascular injury. In conclusion, we report
uFg as a sensitive and specific translational biomarker for tubulovascular inflammation
in the kidney providing mechanistic information about the cause of AKI.
745 METABOLOMICS ANALYSIS SUGGESTS ROLE OF 3-
INDOXYL SULFATE IN CENTRAL NERVOUS SYSTEM
TOXICITY AND AN EARLY INDICATOR OF
CHEMICALLY-INDUCED RENAL FAILURE.
M. V. Milburn 1 , D. Alexander 1 , J. R. Zgoda-Pols 2 , S. Chowdhury 2 , M. Wirth 2
and K. B. Alton 2 . 1 Metabolon, Durham, NC and 2 Department of Drug Metabolism
and Pharmacokinetics, Merck Research Laboratories, Kenilworth, NJ. Sponsor: G.
Vansant.
An investigative renal toxicity study was conducted with Compound A using LC-
MS and GC-MS metabolomics to identify small biomarkers of acute renal failure
(ARF) that could aid in a better mechanistic understanding induced ARF in mice.
The metabolomics study revealed 3-Indoxyl sulfate3 (3IS) as the most sensitive
marker of renal toxicity. 3IS is a known renal toxin that accumulates in blood of
uremic animals or patients due to a decreased or absent urinary excretion of 3IS
during renal failure. The accumulation of uremic toxins such as 3IS in blood and
tissues can also cause multiple physiological changes involving central nervous system
(CNS) dysfunction, such as uremic encephalopathy. Compound B, a close
structural analogue of Compound A, was used as a negative control since it did not
cause ARF in mice and cisplatin (CDDP) was used as a positive control. An LC-
MS-based bioanalytical assay for the determination of 3IS in mouse matrices was
also developed. Following treatment with nephrotoxicants (Compound A or
CDDP), 3IS levels were markedly increased in murine plasma and brain, thereby
contributing to renal- and CNS-related toxicities. Furthermore, as expected urinary
excretion of 3IS appeared to be absent in those animals due to compromised renal
function. Thus, these data suggest that 3IS could potentially serve as a marker of
renal and CNS toxicities during drug-induced ARF in mice. Furthermore, this
comprehensive approach that includes untargeted metabolomic and targeted bioanalytical
sample analyses could be used to investigate toxicity of other compounds
that pose preclinical or clinical development challenges in a pharmaceutical setting.
746 SHORT-TERM TOXICITY OF MELAMINE AND ITS
MIXTURE WITH CYANURIC ACID IN F344 NEONATAL
RATS AND SEARCHING FOR NOVEL KIDNEY
BIOMARKERS.
H. Kang 1 , Y. Park 1 , S. Jeong 2 , J. Seo 1 , Y. Jean 1 , J. Kang 1 , H. Shin 1 and S. Son 1 .
1 Veterinary Toxicology and Chemistry, National Veterinary Research and Quarantine
Service, Anyang, Republic of Korea and 2 GLP Research Center, Hoseo University,
Asan, Republic of Korea.
This study was performed to compare the systemic toxicity between Mel alone and
Me+Cya mixture treated to neonatal F344 rats. 1-day-old male and female F344
rats were administered with Mel (0, 10, 35, and 118 mg/kg bw), Cya (150 mg/kg
bw), and Mel+Cya (0.35+0.35, 1.0+1.0, and 3.5+3.5 mg/kg bw) via gavage once
per day for 4 weeks. Clinical signs, pathological findings, clinical chemistry and
urine test were examined after the treatment. Kidney toxicity biomarkers (KIM-1,
TIMP-1, VGEF, clusterin, NGAL, osteopontin, β2m, GST-α, calbindin and cystatin
C) in serum and urine were analyzed. While there were no gloss pathological
findings in all of treatment groups, relative weight organs of thyroid glands and epididymis
were significantly decreased at Mel+Cya (1.0+1.0 and 3.5+3.5). Serum
total protein and albumin were increased by Mel+Cya (0.35+0.35, 1.0+1.0, and
3.5+3.5). Serum uric acid was decreased by Mel (10, 35, and 118) and Cya. Round
shaped and greenish dark-brown colored crystals were detected in urine sediment at
Mel+Cya (3.5+3.5). Also, polygonal and translucent crystals were found in rats
treated with Mel treatment (118). TIMP-1 in serum was decreased in male and female
rats by Mel+Cya (3.5+3.5). Cystatin C in serum was increased in female rats
by Mel+Cya (3.5+3.5). Osteopontin in serum was decreased in male rats by
Mel+Cya (3.5+3.5). Urinary TIMP-1 and clusterin increased in female rats by Cya
(150) and Mel+Cya (3.5+3.5), respectively. The NOAEL of Mel+Cya is estimated
as 0.35 mg/kg bw/day based on the relative weight organs of thyroid gland and epididymis.
The threshold level of renal toxicity in terms of urinary crystal formation
in Mel+Cya could be between 1.0 mg/kg and 3.5 mg/kg bw/day while in Mel treats
could be between 35 and 118 mg/kg. Conclusively, this study supports that Mel
mixture could also show more toxicity especially in male neonate rats than Mel or
Cya alone.
747 TRANSCRIPTION LEVELS OF BIOMARKERS OF
ACUTE RENAL INJURY IN F344 RATS CO-EXPOSED TO
MELAMINE AND CYANURIC ACID FOR SEVEN DAYS.
L. Camacho, K. Kelly and G. Gamboa da Costa. Division of Biochemical
Toxicology, National Center for Toxicological Research/U.S. FDA, Jefferson, AR.
The intentional adulteration of pet food ingredients with melamine and its derivatives,
including cyanuric acid, was responsible for the renal failure and death of a
substantial number of cats and dogs in the USA. Experimental evidence demonstrates
that the co-exposure to low levels of both compounds elicits renal toxicity
due to the formation of melamine cyanurate crystals in the kidney nephrons. In this
work, we investigated if a co-exposure to melamine and cyanuric acid in rats leads
to alterations in the gene expression of proteins (KIM1, TIMP1, clusterin, osteopontin,
and NGAL) that have been proposed as urinary biomarkers for acute kidney
injury. Male and female F344 rats were fed NIH41 diet supplemented with 0,
69, 229, or 694 ppm each of melamine and cyanuric acid, which resulted in an exposure
of 0, 8.6, 17.6, and 29.8 mg/kg bw/day melamine and cyanuric.
Histopathology and clinical chemistry examination indicated marked toxicity in
the animals exposed to the two highest doses (17.6 and 29.8 mg/kg bw/day) but
not at the lowest dose (8.6 mg/kg bw/day). Quantitative PCR analysis of kidney tissue
indicated increased expression of genes coding for KIM1, TIMP1, clusterin,
and NGAL relative to the control in animals co-exposed to 17.6 and 29.8 mg/kg
bw/day melamine and cyanuric acid. These data suggest that the change in expression
of these genes may constitute an endpoint to assess combined toxicity of
melamine and cyanuric acid in rat kidneys. Funded in part by NTP IAG 224-07-
0007 between the FDA/NCTR and the NIEHS/NTP.
748 THE USE OF FLOW CYTOMETRY TO STUDY
MECHANISMS OF MITOCHONDRIAL BIOGENESIS IN
RENAL CELLS.
V. Kale and R. Schnellmann. Department of Pharmaceutical and Biomedical
Sciences, Medical University of South Carolina, Charleston, SC.
Mitochondria play an important role in the outcome of toxicant exposures and ischemia
reperfusion injury. Mitochondrial biogenesis is tightly regulated by mitochondrial
and nuclear transcriptional factors and results in increased mitochondrial
mass/number. We treated renal proximal tubular cells (RPTC) with three chemicals
that induce mitochondrial biogenesis, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane
(DOI, 5-HT2 agonist); SRT1720 (SIRT1 activator); and metformin
(AMPK activator). We then isolated mitochondria and determined mitochondrial
membrane potential (TMRM), cardiolipin (NAO) content, and relative size (side
scatter, SSC) using flow cytometry. Among the three chemicals tested, DOI (10
μM) increased NAO (16±7-%), and TMRM (19±8-%) staining, and decreased
mitochondrial size and complexity (indicated by an increase in the geometric mean
of SSC (289±55, 575±112)) over vehicle controls after 24 h treatment. Metformin
(1 mM) and SRT1720 (10 μM) treatment of RPTC did not alter NAO staining or
TMRM staining. However, SRT1720 decreased the geometric mean for SSC
(280±21, 196±33) indicating increased mitochondrial size and decreased in the
complexity. Mitochondrial biogenesis was confirmed in each case by demonstrating
that mitochondrial complex proteins (ATPsynthase β & NDUFB8), mitochondrial
copy number and RPTC respiration increased. In conclusion, flow cytometry is a
useful tool to study individual mitochondria following chemical exposures and mitochondrial
biogenesis. Although all chemicals caused mitochondrial biogenesis
only DOI increased cardiolipin staining and membrane potential suggesting that
different inducers of mitochondrial biogenesis cause different mitochondrial phenotypes.
Funding: NIGMS (GM084147).
749 OXIDANT INJURY INDUCES RAPID DEGRADATION
OF MITOCHONDRIAL CALPAIN 10 IN RENAL
PROXIMAL TUBULE CELLS.
M. A. Smith and R. G. Schnellmann. Pharmaceutical Sciences, Medical University
of South Carolina, Charleston, SC.
Calpain 10, which is found in the cytosol and the mitochondrial matrix, has been
shown to cleave proteins in the electron transport chain and ATP synthesis.
Previously, we showed that knockdown of calpain 10 causes renal proximal tubule
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