TOXICOLOGICAL SCIENCES 69, 424 – 432 (2002)
Copyright © 2002 by the Society of Toxicology
Cellular Toxicity of Hydrazine in Primary Rat Hepatocytes
Saber M. Hussain* ,1 and John M. Frazier†
*ManTech Environmental Technology, Inc., Dayton, Ohio 45437; and †Operational Toxicology Branch (AFRL/HEST),
Air Force Research Laboratory, Wright-Patterson Air Force Base, Ohio 45433-7400
Received January 9, 2002; accepted June 10, 2002
Hydrazine (HzN) is an aircraft fuel and propellant used by the
U.S. Air Force. The current study was undertaken to evaluate the
acute toxicity of HzN in primary rat hepatocytes in vitro with
reference to oxidative stress. The effects of short-term exposure (4
h) of hepatocytes to HzN were investigated with reference to
viability, mitochondrial function, and biomarkers of oxidative
stress. The viability data showed an increase in lactate dehydrogenase leakage and a decrease in mitochondrial activity with
increasing concentration of HzN. The results of studies of oxidative stress biomarkers showed a depletion of reduced glutathione
(GSH) and an increase in oxidized GSH, increased reactive oxygen
species generation, lipid peroxidation, and reduced catalase activity. Furthermore, depletion of GSH and catalase activity in hepatocytes by buthionine sulfoximine and 3-amino triazole, respectively, prior to exposure to HzN, increased its toxicity. The results
suggest that acute HzN-induced cytotoxicity in rat hepatocytes is
likely to be mediated through oxidative stress.
Key Words: hydrazine; in vitro; hepatocytes; oxidative stress;
catalase; glutathione; reactive oxygen species.
Hydrazine (N 2H 4; HzN), a simple diamine and powerful
reducing agent, has been used as a fuel and propellant in
aircraft by the U.S. Air Force. Anhydrous HzN (MP 2°C, BP
113.5°C) is an oily hygroscopic liquid that fumes in air, releasing a penetrating odor resembling ammonia. Because of its
desirable physical characteristics, such as high boiling point,
thermal stability, and spontaneous ignition with strong oxidizing agents, it is an excellent choice for rocket and jet propellants. Various forms of HzN derivatives, e.g., Ultra Pure™,
monomethyl HzN, and unsymmetrical dimethyl HzN, are used
in both mono- and bipropellant systems. A mixture of 70%
HzN and 30% water (H-70) is used in the F-16 fighter aircraft
emergency power unit. The National Aeronautics and Space
Administration also uses HzN as propellants on the space
shuttle and several satellite systems. Besides their utilization as
propellants, HzN derivatives are also used in industry to pro1
To whom correspondence should be addressed at Air Force Research
Laboratory, AFRL/HEST, ManTech Environmental Technology, Inc., P.O.
Box 31009, Dayton, OH 45437-0009. Fax: (937) 258-2197. E-mail:
[email protected].
duce plastic blowing agents, dyes, herbicides, and drugs (Blair
et al., 1985; Timbrell and Harland, 1979).
The primary source of exposure to U.S. Department of
Defense personnel occurs from the fueling of rockets and
aircraft propulsion systems. To protect personnel, a full-body
protection suit is required when working with HzN propellants.
Because of the highly toxic nature of HzN, novel candidate
chemicals are being investigated by the U.S. Air Force Research Laboratories as possible replacements that would have
equivalent performance characteristics but less toxicity (Hussain and Frazier, 2001). Therefore, understanding the biochemical basis of HzN toxicity is an essential starting point for
developing replacement chemicals.
Previous reports have identified several toxic effects associated with exposure to HzN compounds, e.g., liver damage,
hyperglycemia, neurodegeneration, and cancer (Kenyon et al.,
1999; Moloney and Prough, 1983; Petersen et al., 1970; Wald
et al., 1984). Experimentally in rats, HzN causes depletion of
glutathione (GSH) and the accumulation of triglycerides in the
liver (Jenner and Timbrell, 1994). In addition, HzN interferes
with the urea cycle of the rat liver (Roberge et al., 1971). HzN
has been reported to induce methylation of DNA (Bosan et al.,
1987). HzN exposure leads to adenosine triphosphate (ATP)
depletion and megamitochondria formation in vivo (Kerai and
Timbrell, 1997; Preece et al., 1990; Wakabayashi et al., 2000).
HzN inhibits the mitochondrial enzyme succinate dehydrogenase (Ghatineh et al., 1992), which subsequently reduces mitochondrial function. Further, HzN produces toxicity by interfering with a number of metabolic processes such as
gluconeogenesis (Kleineke et al., 1979) and glutamine synthetase (Kaneo et al., 1984; Noda et al., 1987; Sendo et al.,
1984; Willis, 1966).
Oxidative stress plays a role in the mechanisms of toxicity of
a number of compounds, whether by production of free radicals or by depletion of cellular antioxidant capacity. Cellular
integrity is affected by oxidative stress when the production of
reactive oxidants overwhelms antioxidant defense mechanisms
(Halliwell et al., 1992; Yu, 1994). The metabolism of HzN and
its derivatives is thought to involve the production of free
radicals that may induce cellular toxicity either by covalent
binding to tissue macromolecules or by initiating an autoxidative process such as lipid peroxidation in vivo (Choudhary and
424
CELLULAR TOXICITY OF HYDRAZINE
Hansen, 1998; Preece and Timbrell, 1989). Evidence for the
production of radicals, including methyl, acetyl, hydroxyl, and
hydrogen radicals, has been observed during the metabolism of
HzN (Ito et al., 1992; Noda et al., 1988; Runge-Morris et al.,
1988; Sinha, 1987). Thus, studies show that multiple pathways,
both enzymatic and nonenzymatic, appear to be involved in
free radical generation. Free radicals have been implicated in
protein (hemoglobin) damage associated with HzN in human
erythrocytes, suggesting that free radicals may be involved in
the anemic effects of HzN observed in animals in vivo (RungeMorris et al., 1988). Therefore, the formation of free radicals
during the metabolism of HzN may be important to the mechanism of action of HzN toxicity.
It is known from the literature that HzN interferes in a broad
range of physiological reactions. However, a complete toxicological profile and the mechanism of HzN toxicity are not yet
fully understood. No studies are available on the acute lethality
of HzN in vivo. Previous in vitro studies are based on 24-h
exposures at minimally lethal doses of HzN. Therefore, the
objective of this study was to investigate the acute cytotoxicity
of HzN exposure in primary rat hepatocytes following short
(4-h) exposures. The present study describes the acute cytolethality of HzN, and some aspects of its mechanism of toxicity
in primary rat hepatocytes with reference to oxidative stress.
425
seeded in either 96-well (4 ⫻ 10 4 cells/well) or 6-well (1.0 ⫻ 10 6 cells/well)
culture plates previously coated with rat tail collagen (1.0 ␮g/well or 25
␮g/well, respectively). After 4-h incubation in a 5% CO 2 incubator at 37°C to
allow for attachment, hepatocytes were re-fed with fresh Chee culture medium
lacking dexamethasone. Hepatocytes were cultured for an additional 20 h
before treatment.
Treatment. In all studies, primary rat hepatocytes were treated for 4 h with
various concentrations of HzN dissolved in Chee culture media. At the end of
the exposure period, toxicity end points were evaluated.
LDH leakage. Lactose dehydrogenase (LDH) leakage was assessed by
spectrophotometrically measuring the oxidation of NADH at 340 nm in both
the cells and media, as described by Moldeus et al., (1978). The percent of
activity in the media was then calculated by dividing the amount of activity in
the media by the total activity (medium plus cell lysate).
Mitochondrial function. Mitochondrial function was determined spectrophotometrically by measuring the reduction of the tetrazolium salt MTT to
formazan by succinic dehydrogenase as previously described (Carmichael et
al., 1987).
Reduced and oxidized GSH. Reduced GSH and oxidized glutathione
(GSSG) were measured in 96-well plates using a SpectraMAX Plus 190
microplate reader (Molecular Devices, Sunnyvale, CA.) according to the
procedures described in the Glutathione Assay Kit (Cayman Chemical Company, Ann Arbor, MI). The assay is based on the enzymatic recycling method,
using glutathione reductase (GR) and 5,5⬘-dithiobis-2-nitrobenzoic acid (Ellman’s reagent; Tietze, 1969). Data are reported as percent of control.
MATERIALS AND METHODS
Reactive oxygen species (ROS) generation. ROS generation was determined using the method described by Wang and Joseph (1999). Fluorescence
was detected using an excitation wavelength of 485 nm and an emission
wavelength of 530 nm. Data are reported as fold increase in fluorescence
intensity relative to control.
Chemicals. Collagenase was obtained from Boehringer-Mannheim Biochemicals (Indianapolis, IN), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ␤-nicotinamide-adenine dinucleotide-reduced
(NADH), 2⬘-7⬘-dichlorodihydrofluorescein diacetate (DCFH-DA), reduced
GSH, rhodamine 123, buthionine-[S,R]-sulfoximine (BSO), insulin/transferrin/sodium selenite (ITS) additive, gentamicin, and dexamethasone were
purchased from Sigma Chemical Company (St. Louis, MO). Chee media was
obtained from Gibco (Grand Island, NY). Hydrazinium nitrate was supplied
from the Propulsion Directorate of the U.S. Air Force Research Laboratory,
Edwards Air Force Base, CA.
Lipid peroxidation. The extent of lipid peroxidation in control or HzNexposed hepatocytes was determined by measuring the thiobarbituric acid
reactive substances (TBARS). TBARS were determined according to the
procedures of Ohkawa et al. (1979) with minor modifications. At the end of
exposure, cells were washed and scraped into 1 ml phosphate-buffered saline
(PBS). This was followed by addition of 100 ␮l of 10% sodium dodecyl sulfate
for solubilization. Then 650 ␮l of 0.5% thiobarbituric acid in 20% (v/v) glacial
acetic acid (pH 3.5) were added and incubated at 80°C for 30 min. After
incubation, the samples were cooled and the absorbance was measured at 532
nm in a SpectraMAX Plus 190 microplate reader (Molecular Devices, Sunnyvale, CA). Data are expressed as nmol TBARS per mg cellular protein.
Animals. Male Fischer 344 rats (225–300 g) were obtained from Charles
River Laboratory (Raleigh, NC). Rats were anesthetized with 1 ml/kg of a
mixture of ketamine (70 mg/ml; Parke-Davis, Morris Plains, NJ) and xylazine
(6 mg/ml; Mobay Corp., Shawnee, KS) prior to undergoing liver perfusion. All
animals used in this study were handled in accordance with the principles
stated in the Guide for the Care and Use of Laboratory Animals, Institute of
Laboratory Animal Resources, and the Animal Welfare Act of 1966, as
amended (National Research Council, National Academy Press, 1996).
Liver perfusion, hepatocytes enrichment, and culture. Rat livers were
perfused, and hepatocytes were isolated and enriched by the two-step Seglen
procedure (Seglen, 1976) with minor modifications as previously described
(DelRaso and Frazier, 1999). For all perfusions, Chee medium (pH 7.2) was
supplemented with 10 mM HEPES. Washout medium was further supplemented with heparin (2.0 U/ml) and EGTA (0.5 mM), and digestion medium
was supplemented with 500 mg/l collagenase. Viable primary rat hepatocytes
were enriched by low-speed centrifugation (500 ⫻ g) for 3 min. Typically, the
yield of isolated hepatocytes was from 300 to 400 million cells, with viability
ranging from 85 to 95% as determined by trypan blue dye exclusion. For cell
culture studies, suspensions of primary hepatocytes were adjusted to a cell
density of 1.0 ⫻ 10 6 cell/ml in Chee culture medium (pH 7.2) supplemented
with HEPES (10 mM), insulin/transferrin/sodium selenite (5 mg/l, 5 mg/l, 5
␮g/l), gentamicin (50 mg/l), and dexamethasone (0.4 mg/ml). Cells were
Determination of mitochondrial membrane potential. Mitochondrial
membrane potential was determined by the uptake of rhodamine 123 according
to the method of Wu et al. (1990). After treatment, hepatocytes cultured in
96-well plates were washed with PBS and incubated with 10 ␮g/ml rhodamine
123 (Molecular Probes, Inc. Eugene, OR) in Chee medium for 30 min at 37°C.
After further washing with PBS, the hepatocytes were incubated in Chee media
for 30 min. After removing the media, 0.2 ml ethanol/water solution (1 part
ethanol:1 part water) was added to extract the dye retained by the cells.
Fluorescence was measured with a SpectraMAX Gemini-XS fluorescence
plate reader (Molecular Devices, Sunnyvale, CA) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Data are expressed
as percent of control uptake.
Antioxidant enzymes. Treated hepatocytes were washed with PBS and
scraped into 1 ml PBS. A cell pellet was obtained by centrifugation at 500 ⫻
g for 5 min. The pellet was resuspended in potassium phosphate buffer (pH
7.2), placed on ice, and sonicated three times (10 s each) with an Ultrasonic
Homogenizer (Cole-Pharmer Instrument Company, Chicago, IL). The resulting homogenates were centrifuged for 30 min at 18,000 ⫻ g (4°C) and the
supernatant was collected in a SpectraMAX Plus 190 microplate reader (Molecular Devices, Sunnyvale, CA) to measure enzyme activities. Catalase activity was assayed in the supernatant by the method of Aebi (1984), which
involves monitoring the disappearance of hydrogen peroxide (H 2O 2) at 240
426
HUSSAIN AND FRAZIER
nm. The enzymatic activity was expressed in Units/mg cellular protein. Glutathione peroxidase (GPx) activity in the supernatant was estimated according
to the method of Flohe and Gunzler (1984). GR was measured according to the
method of Carlberg and Mannervik (1985). GPx and GR activities were
expressed in mUnits/mg cellular protein (one mUnit of activity represents one
nmol NADPH oxidized per minute).
Determination of protein concentration. Total protein concentration in
cell lysate was determined using an ESL Protein Assay Kit according to
manufacturer’s instructions (Boehringer-Mannheim Biochemicals, Indianapolis, IN). This method is an optimized procedure that combines the biuret
reaction and the copper (I)-bathocuproine chelate reaction, as described by
Matsushita et al. (1993).
Statistical analysis. The data are expressed as means ⫾ SD of three
independent experiments with hepatocytes from three different rats. The data
were subjected to statistical analysis by one-way analysis of variance
(ANOVA) followed by Dunnett’s method for multiple comparisons. A value of
p ⬍ 0.05 was considered significant. The SigmaStat for Windows version 2.03
software is used for the statistical analysis.
RESULTS
Initially, a range-finding study was conducted to determine
the HzN exposure conditions that resulted in acute cytolethality. In these studies, hepatocytes were exposed to HzN at
concentrations ranging from 1 to 150 mM for 1, 2, 4, 6, 12, or
24 h and assayed immediately for cytotoxicity using LDH
leakage and MTT reduction as end points (Figs. 1A and 1B).
Membrane damage that results in LDH leakage is generally
considered irreversible. Therefore, LDH leakage was selected
as a biomarker for cellular viability. The results for LDH
leakage indicate that a 1- or 2-h exposure did not result in any
cytotoxicity, whereas the 4-, 5-, 6-, 12-, and 24-h exposure
induced significant concentration-dependant cytotoxicity (Fig.
1A). The MTT assay was used to assess the concurrent effects
of HzN on mitochondrial function of rat hepatocytes. MTT
results, as displayed in Figure 1B, showed moderate decreases
in MTT reduction at 1 and 2 h, with significant toxicity at 4, 5,
6, 12, and 24 h. The estimated EC 50s for LDH leakage and
MTT for each exposure time are shown in Table 1. The
concentration-exposure time product (CT values) for a specific
level of effect, calculated by multiplying the EC 50 by the
exposure time, represents the area under the exposure curve
giving a 50% response. The data in Table 1 indicate that the CT
product is not constant, but tends to decrease with increasing
exposure time up to 12 h and then increase for the 24-h
continuous exposure duration.
From these initial experiments, significant cytotoxicity was
apparent for the 4-h exposures. Therefore, the 4-h exposure
period was selected for short-term acute cytotoxicity studies of
HzN. Figure 1A indicates that a 4-h exposure to HzN produced
a dose-dependent increase in LDH leakage with 70 – 80% LDH
leakage at 150 mM HzN. The lowest concentration resulting in
a statistically significant increase in LDH leakage was 25 mM,
and the EC 50 was calculated to be 80 mM. Continuous HzN
exposure for 4 h resulted in a concentration-dependent decrease in mitochondrial function with an EC 50 of 30 mM (Fig.
FIG. 1. (A) Effect of HzN on LDH leakage of rat hepatocytes. Primary
hepatocytes were treated with different concentrations of HzN for 1–24 h. At
the end of the incubation period, the percentage of LDH released into the
media was measured by spectrophotometric analysis as described in Materials
and Methods. (B) Effect of HzN on mitochondrial function (MTT) of rat
hepatocytes. Primary hepatocytes were treated with different concentrations of
HzN for 1–24 h. At the end of the incubation period, mitochondrial function
was determined by the MTT reduction, as described in Materials and Methods.
*Statistically significant difference compared with controls (p ⬍ 0.05).
1B). A statistically significant decrease in MTT reduction was
observed at 25 mM, whereas the highest exposure (150 mM)
resulted in a 90% reduction in mitochondrial function after 4-h
exposure.
DCFH-DA is widely used to measure ROS generation in
cells. ROS generation following 4-h exposure to HzN is shown
in Figure 2A. The levels of ROS in hepatocytes increased in a
concentration-dependent manner and were statistically increased (p ⬍ 0.05) at the lowest concentration studied (25
mM). HzN treatment at 100 and 150 mM resulted in an
approximately 8-fold increase in ROS over control levels.
Measurement of lipid peroxidation is used to investigate the
process of cellular damage induced by free radicals. There was
CELLULAR TOXICITY OF HYDRAZINE
TABLE 1
EC 50 and CT Values for LDH and MTT
EC 50 (mM)
Exposure time (h)
1
2
4
6
12
24
CT (mM-h)
LDH
MTT
LDH
MTT
⬎150
⬎150
78
22
7.2
6
⬎150
93
37
16.8
6.6
5.8
—
—
312
132
86.4
144
—
186
148
100.8
111
139.2
Note. EC 50 values represent effective concentrations of HzN that increase
LDH leakage to 50% or decrease MTT reduction by 50%. CT values (EC 50 ⫻
exposure time) represent the area under the exposure curve; —, CT values
could not be estimated.
no significant increase in the level of TBARS at 10, 25, and 50
mM HzN (Fig. 2B). However, the variability between the
responses of the three separate hepatocyte preparations, as
indicated by the standard deviation, increased significantly at
the 50 mM concentration. At the higher exposure concentrations (100 and 150 mM), TBARS levels in cells were significantly elevated, approximately 2-fold, over control levels.
These results indicate lipid peroxidation is observed at higher
HzN exposures.
The effect of HzN on mitochondrial membrane potential
(MMP) was evaluated in primary rat hepatocytes (Fig. 3).
Hepatocytes were exposed to 10, 25, 50, 100, and 150 mM
HzN for 4 h and immediately assayed for rhodamine 123
uptake. The results showed that there was a significant decrease of MMP at 50, 100, and 150 mM HzN. The reduction of
MMP that was observed following exposure to 100 mM HzN
was 55% at 4-h exposures. These results revealed that exposure
to concentrations of HzN greater than 50 mM significantly
affected mitochondrial function, as indicated by a decrease in
MMP in primary hepatocytes.
GSH is a ubiquitous sulfhydryl-containing molecule in cells
that is responsible for maintaining cellular oxidation-reduction
homeostasis. Changes in GSH homeostasis can be monitored
as an indication of cell damage. GSH and GSSG levels were
measured in control and HzN-exposed cells after 4-h exposure
(Figs. 4A and 4B, respectively). A significant depletion of
GSH (70%) was observed at the lowest HzN concentration
studied (25 mM) relative to controls (55.8 ⫾ 5. 6 nmol
GSH/mg cellular protein). Additional decreases in GSH were
observed at 50, 100, and 150 mM exposures (Fig. 4A). Increased GSSG levels were found at all exposure concentrations
(25–150 mM) relative to controls (7.5 ⫾ 1.2 nmol/mg cellular
protein). The 25-mM HzN treatment resulted in a 65% increase
in GSSG, with no further increase in GSSG at high exposures
(Fig. 4B). Overall, the data demonstrated a significant depletion of GSH levels in HzN-exposed cells, with an increase of
GSSG levels at all doses tested.
427
To further investigate the potential role of oxidative stress in
the mechanism of toxicity of HzN, the effect of modulating
GSH levels in rat hepatocytes on cytotoxicity was studied. To
deplete intracellular GSH, cells were incubated with various
concentrations of L-buthionine-SR-sulfoximine (BSO), a selective inhibitor of ␥-glutamylcysteine synthetase, during the 20-h
recovery period between hepatocyte isolation and HzN treatment. Preliminary studies indicated a significant depletion (⬎
90%) of GSH at BSO concentrations of 0.2, 0.5, and 1 mM
(data not shown) without any effect on cell viability, as determined by the MTT assay. The lowest dose of BSO (0.2 mM)
was selected for GSH depletion studies. Cells pretreated with
0.2 mM BSO for 20 h were exposed to HzN for 4 h. The MTT
FIG. 2. (A) Effect of HzN on ROS generation in rat hepatocytes. Primary
hepatocytes were incubated with DCFH-DA for 30 min. After DCFH-DAcontaining medium was removed, the cells were washed and treated with HzN
in Chee media for 4 h. At the end of exposure, fluorescence was measured, as
described in Materials and Methods section, and the intensity of fluorescence
expressed in fold increase in treated cells with respect to control. (B) Effect of
HzN on lipid peroxidation in rat hepatocytes. Primary hepatocytes were treated
with different concentrations of HzN for 4 h. At the end of the incubation
period, cells were collected and TBARS were determined spectrophotometrically, as described in Materials and Methods section. Data are expressed as
means ⫾ SD of three independent experiments with hepatocytes from three
different rats. *Statistically significant difference compared with controls (p ⬍
0.05).
428
HUSSAIN AND FRAZIER
sensitivity to HzN over the entire range of HzN concentrations
tested compared with cells with normal catalase activity. In
spite of the fact that the extent of the effect of catalase inhibition on MTT reduction was less than the effect of GSH
depletion, these results clearly demonstrate that inhibiting catalase activity also has a modulating effect on HzN toxicity.
DISCUSSION
FIG. 3. Effect of HzN on MMP in rat hepatocytes. Primary hepatocytes
cultured in 96-well plates were exposed to HzN for 4 h. Immediately after
treatment, cells were assayed for rhodamine 123 uptake, as described in the
Materials and Methods section. Data are expressed as means ⫾ SD of three
independent experiments with hepatocytes from three different rats. *Statistically significant difference compared with controls (p ⬍ 0.05).
assay was performed to examine mitochondrial function in
GSH-depleted cells (Fig. 5). GSH-depleted cells showed a
significant increase in sensitivity to HzN compared with normal hepatocytes. At the 25 mM HzN exposure, there was about
an 80% decrease in MTT reduction activity in GSH-depleted
cells, with the EC 50 being less than 25 mM (compared with 65
mM in GSH-competent cells). These results clearly demonstrate that GSH depletion in rat hepatocytes increased sensitivity to HzN.
To assess the potential role of antioxidant enzymes in HzN
toxicity, catalase, GPx, and GR activities were analyzed in
control cells and cells exposed to HzN for 4 h. Figure 6 shows
a dose-dependent inhibition of catalase activity. There was a
significant reduction (approximately 50%) in catalase activity
at 25 mM, with a further reduction to 90% at the 150 mM HzN
treatment. Activities of GSH-dependent enzymes GPx and GR
were slightly increased (statistically significant) in HzN-treated
cells at 50 mM concentrations, but no consistent response
pattern was observed. The results indicate that among antioxidant enzymes investigated, HzN appears to inhibit catalase
activity, whereas GPx and GR are relatively unaffected.
The effect of reduced catalase activity on HzN toxicity was
also evaluated in rat hepatocytes. Amino triazole (AT) inhibits
catalase activity, making cells more susceptible to certain toxic
chemicals (Hussain et al., 1999). Hepatocytes were exposed to
5 mM AT during the 20-h recovery period between hepatocyte
isolation and HzN treatment. Treatment with 5 mM AT resulted in complete inhibition of catalase activity (100% inhibition), with a concurrent increase in MTT activity of about
20%. Cells pretreated with AT were exposed to different
concentrations of HzN for 4 h. Immediately after exposure, the
MTT assay was performed to examine mitochondrial function
in HzN-treated catalase inhibited cells. As presented in Figure
7, cells in which catalase was inhibited by AT showed a greater
Risk characterization of the acute toxicity of a chemical
must consider two major components, lethality and morbidity,
following a single exposure or several repeated exposures in
rapid succession to the chemical of concern. In general, lethality (catastrophic systems failure) is a consequence of either
target organ failure or failure of higher level systems control
processes. In the case of HzN, the available literature suggests
FIG. 4. Effect of HzN on (A) GSH and (B) on GSSG levels in rat
hepatocytes. Primary hepatocytes were treated with different concentrations of
HzN for 4 h. At the end of the exposure, cells were washed with PBS, and GSH
(control: 55.8 ⫾ 5.6 ␮mol/mg protein) and GSSG (control 7.5 ⫾ 1.2 ␮mol/mg
protein) levels were measured, as described in the Materials and Methods
section. Data are expressed as means ⫾ SD of three independent experiments
with hepatocytes from three different rats. *Statistically significant difference
compared with controls (p ⬍ 0.05).
CELLULAR TOXICITY OF HYDRAZINE
FIG. 5. Effect of GSH depletion on HzN toxicity in rat hepatocytes.
Primary hepatocytes were treated during the recovery period with 0.2 mM
BSO in Chee media. Following BSO pretreatment, cells were washed with
PBS and exposed to different concentrations of HzN for 4 h. Control cells with
no BSO treatment were exposed to HzN concurrently at the same concentrations. At the end of the exposure period, mitochondrial function was evaluated
by MTT reduction, as described in the Materials and Methods section. Data are
expressed as means ⫾ SD of three independent experiments with hepatocytes
from three different rats. *Statistically significant difference compared with
controls (p ⬍ 0.05).
that acute liver failure is a likely candidate for the etiology of
acute lethality. The purpose of this investigation was to elucidate potential mechanisms involved in acute lethality of HzN.
Hepatocytes were exposed to concentrations of HzN between
FIG. 6. Effect of HzN on antioxidant enzymes in rat hepatocytes. Primary
hepatocytes were treated with different concentrations of HzN for 4 h. At the
end of the exposure, cells were washed with PBS and a cell lysate prepared as
described in the Materials and Methods section. Catalase activity (control:
673.86 ⫾ 44.3 U/mg protein) was determined spectrophotometrically by
measuring the disappearance of H 2O 2 at 240 nm, as described in the Materials
and Methods section. GPx activity (control: 212.21 ⫾ 10.23 mU/mg protein)
was determined spectrophotometrically by measuring the oxidation of NADPH
at 340 nm, as described in the Materials and Methods section. GR activity
(control: 191.85 ⫾ 8.6 mU/mg protein) was determined spectrophotometrically by measuring the oxidation of NADPH at 340 nm, as described in the
Materials and Methods section. Data are expressed as means ⫾ SD of three
independent experiments with hepatocytes from three different rats. *Statistically significant difference compared with controls (p ⬍ 0.05).
429
FIG. 7. Effect of catalase inhibition on HzN toxicity in rat hepatocytes.
Primary hepatocytes were treated during the recovery period with 5 mM AT in
Chee media. Following AT pretreatment, cells were washed with PBS and
exposed to different concentrations of HzN for 4 h. Control cells with no AT
treatment were exposed to HzN concurrently at the same concentrations. At the
end of the exposure period, mitochondrial function was determined spectrophotometrically by measuring the degree of MTT reduction, as described in the
Materials and Methods section. Data are expressed as means ⫾ SD of three
independent experiments with hepatocytes from three different rats. *Statistically significant difference compared with controls (p ⬍ 0.05).
10 and 150 mM, based on our range-finding studies, to elicit
acute cytotoxic responses in the 4-h exposure protocol employed. The concentrations of HzN used in these studies are
higher and the exposure time is shorter (4 h) when biochemical
end points are observed relative to previous studies. These
doses and times are relevant to single exposures to HzN in an
accidental exposure scenario.
To put this into perspective, Preece et al. (1992) investigated
the hepatotoxic effects of a single oral dose of HzN in rats. At
the highest dose studied (81 mg/kg), they observed minor liver
pathology, i.e., intracellular fat droplets in two of three rats, but
no deaths at 4 days postexposure. The maximum plasma concentration observed following the 81 mg/kg dose was slightly
over 1 mM (at 90 min postdosing) and the area-under-theplasma-curve (AUC) was roughly 1 mM-h. The lowest concentration found to be cytolethal to isolated rat hepatocytes
after 4 h of exposure in our study, 25 mM, results in an AUC
of about 100 mM-h. Thus, there is an approximately 100-fold
difference in the AUC between accumulation of lipid droplets
in vivo at 4 days and detectable cytolethality in vitro at 4 h. The
two end points are quite different, making a meaningful comparison difficult. However, considering the differences in the
magnitude of toxicity, subclinical/morbidity versus lethality,
and the differences in time frames, 4 days versus 4 h, the
observations on the mechanisms of action of HzN presented
here are probably relevant to the issue of acute lethality.
The results observed for biochemical end points described in
this paper demonstrate that HzN increased LDH leakage and
reduced MTT reduction in a dose-dependent manner over a
wide range of exposure durations. However, mitochondria
appear to be more vulnerable to HzN exposure at shorter
430
HUSSAIN AND FRAZIER
exposure times, as indicated by the differences in EC 50s, e.g.,
MTT reduction (EC 50 [4h]: 37 mM) compared with LDH
leakage (EC 50 [4h]: 78 mM). Although the molecular target for
HzN in the mitochondria is not known with certainty, it appears that mitochondrial dysfunction plays an important a role
in HzN toxicity.
Another interesting observation relating to the time-course
studies is the dependence of the CT product on exposure time.
The fact that the CT product is not constant implies that the
mechanisms of toxicity are highly nonlinear with dose. This
suggests that acute cytolethality may be a consequence of a
different mechanism of action than is operative at lower doses
and longer exposure times.
Cell integrity is affected by oxidative stress when the production of active oxidants overwhelms antioxidant defense
mechanisms. Hepatocytes live in a balance of free radical
production, free radical scavenging, and repair of damage
caused by free radicals (Cai et al., 1995). The addition of HzN
can upset this balance by inducing increased formation of ROS
through mitochondria dysfunction and/or depleting or inhibiting antioxidant systems. The ROS increase following exposure
to any chemical depends on the balance between oxidative and
antioxidant cellular systems. ROS are by-products of biological redox reactions and are involved in various pathological
conditions (Farber et al., 1990). The results here show that
there was a significant increase in ROS at the lowest concentration of HzN studied (25 mM). ROS generation increased
with exposure concentration up to 100 mM, with no further
increase at 150 mM. The apparent lack of increase in ROS at
the higher exposure concentrations may be a consequence of
the leakage of fluorescent product from the cell, as evidence of
significance membrane damage (LDH leakage) was apparent at
the highest concentration. Increased generation of ROS by
HzN is likely to contribute to oxidative stress that may ultimately lead to the observed cytotoxicity (Loft and Poulsen.
1999; Preece and Timbrell, 1989). Another index of oxidative
stress is lipid peroxidation, an important organic biomarker of
oxidative stress induced by reactive free radicals (Duthie,
1993; Kappus, 1987). The toxicity of HzN, as indicated by
LDH release and MTT reduction, is strongly correlated to ROS
generation and lipid peroxidation, indicating induction of
marked oxidative stress in primary culture of rat hepatocytes
following HzN exposure.
Mitochondria are vulnerable targets for toxic injury by a
variety of compounds because of their crucial role in maintaining cellular structure and function via aerobic ATP production.
Our results indicate that the mitochondrial membrane potential
is reduced with increasing concentration of HzN. It has been
suggested that mitochondrial membrane disruption due to altered membrane potential contributes to release of the apoptotic factor cytochrome c. (Kluck et al., 1997; Liu et al., 1996).
Thus, shutdown of mitochondrial function under conditions of
oxidative stress may induce apoptosis at exposure concentrations that do not lead directly to cytolethality.
GSH is a ubiquitous sulfhydryl-containing molecule in cells
that is responsible for maintaining cellular oxidation-reduction
homeostasis (Sies, 1999). GSH protects cells against damage
by scavenging highly reactive free radicals that otherwise
would interact with critical cellular components (Hayes and
McLellan, 1999). Therefore, changes in GSH homeostasis can
be monitored as an indication of cell damage. It is interesting
to note that the lowest concentration of HzN tested (25 mM)
depleted GSH significantly. It is not known how HzN depletes
GSH levels—whether it binds directly to GSH, inhibits enzymes involved in GSH synthesis, or increases GSH consumption in secondary enzymatic reactions. Colvin et al. (1969)
reported that GSH and cysteine adducts have been observed
upon incubation of isopropylhydrazine or acetylhydrazine with
microsomes in the presence of NADPH. Experimentally in
rats, HzN causes depletion of GSH and the accumulation of
triglycerides in the liver (Jenner and Timbrell, 1994). ). It is
likely that GSH depletion may be due to its reaction with HzN
or its metabolites. GSH depletion in primary culture of rat
hepatocytes exposed to HzN is strongly correlated to increased
ROS generation. It has been postulated that the loss of GSH
may compromise cellular antioxidant defenses and lead to the
accumulation of ROS that are generated as by-products of
normal cellular function. Previously, it was shown that the
depression of GSH concentration increased endogeneous ROS
to toxic levels in hepatocytes (Anundi et al., 1979). As demonstrated in our study, depletion of GSH with BSO resulted in
an increased susceptibility to HzN. The EC 50 when normal
hepatocytes were exposed to HzN was 65 mM, and this measure of the toxicity was reduced to 18 mM in GSH-depleted
hepatocytes that was a 4-fold reduction in EC 50 (Fig. 5).
Studies with cultured cells have demonstrated that upon depletion of antioxidant resources such as GSH and catalase,
cells become susceptible to chemicals known to generate ROS
(Hussain et al., 1999; Jones et al., 1978). The results of this
study strongly suggest that GSH is playing a role in protecting
cells from HzN toxicity.
Cellular defenses also include antioxidant enzymes, i.e.,
catalase, GPx, GR, and superoxide dismutase (SOD), that
prevent oxidative damage from ROS. Catalase is one of the
major antioxidant enzymes involved in the detoxification of
H 2O 2, which is produced as a result of the dismutase of
superoxide catalyzed by SOD (Harris, 1992). It is apparent
from the data presented here that inhibition of catalase increases HzN-induced oxidative stress. Studies with isolated
hepatocytes have demonstrated that under conditions of GSH
depletion, catalase functions in the catalysis of H 2O 2 produced
by the cytochrome P450-linked monooxygenase system (Jones
et al., 1978). Thus, under the condition of extreme oxidative
stress, catalase may become important in providing cytoprotection. For example, doxorubicin-induced cardiotoxicity is
suppressed by overexpression of catalase in the heart of transgenic mice (Kang et al., 1986). Experimental studies described
here were conducted to ascertain the effects of HzN on the
CELLULAR TOXICITY OF HYDRAZINE
enzyme activities of catalase, GPx, and GR. The data show that
the activity of catalase decreases markedly in HzN-treated
cells. However, GPx and GR activities remain relatively unaffected. To further evaluate the role of catalase, 3-amino triazole
was used to inhibit the catalase activity in hepatocytes that
were then exposed to HzN. The inhibition of catalase dramatically increased the susceptibility of cells to HzN. These results
support the hypothesis that catalase is a key factor in protecting
cells against HzN toxicity.
In summary, this study has shown that HzN causes increased
generation of ROS, the depletion of GSH, marked inhibition of
catalase activity, increased reduction of MMP, and the increased sensitivity of hepatocytes following GSH depletion
and catalase inhibition. The rapid depletion of GSH by acute
HzN toxicity may impair the cell’s defense against oxidative
stress. There is a strong correlation between GSH depletion
and ROS generation that suggests induction of oxidative stress.
An elevated lipid peroxide profile also is indicative of oxidative stress. In conclusion, our data provide evidence that acute
cytotoxicity of HzN is primarily the result of induction of
oxidative stress.
ACKNOWLEDGMENTS
This work was supported by the U.S. Air Force Office of Scientific Research
(AFOSR) Project (JON# 2312A205) and performed in conjunction with U.S.
Air Force Contract F41624 –96-C-9010 (ManTech/Geo-Centers Joint Venture). We would like to acknowledge Darin Minnick, and TSgts. Gerri Miller
and Michelle Curran for their excellent technical support.
REFERENCES
431
exposure on cadmium cytotoxicity in primary rat hepatocytes. Toxicol.
Methods 9, 97–114.
Duthie, G. G. (1993). Lipid peroxidation. Eur. J. Clin. Nutr. 47, 759 –764.
Farber, J. L., Kyle, M. E., and Coleman, J. B. (1990). Mechanisms of cell
injury by activated oxygen species. Lab. Invest. 62, 670 – 679.
Flohe, L., and Gunzler, W. A. (1984). Assays of glutathione peroxidase.
Methods Enzymol. 105, 114 –121.
Ghatineh, S., Morgan, W., Preece, N. E., and Timbrell, J. A. (1992). A
biochemical and NMR spectroscopic study of hydrazine in the isolated rat
hepatocyte. Arch. Toxicol. 66, 660 – 668.
Harris, E. D. (1992). Regulation of antioxidant enzymes. FASEB J. 6, 2675–
2683.
Halliwell, B., Gutteridge, J. M., and Cross, C. E. (1992). Free radicals,
antioxidants, and human disease: Where are we now? J. Lab. Clin. Med.
119, 598 – 620.
Hayes, J. D., and McLellan, L. I. (1999). Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defense against oxidative
stress. Free Radical Res. 31, 273–300.
Hussain, S., and Frazier, J. (2001). In vitro methods for toxicity assessment of
high energy compounds. Sci. Total Environ. 274, 151–160.
Hussain, S., Hass, B. S., Slikker, W., Jr., and Ali, S. F. (1999). Reduced levels
of catalase activity potentiate MPP ⫹-induced toxicity: comparison between
MN9D cells and CHO cells. Toxicol. Lett. 104, 49 –56.
Ito, K., Yamamoto, K., and Kawanishi, S. (1992). Manganese-mediated oxidative damage of cellular and isolated DNA by isoniazid and related
hydrazines: non-Fenton-type hydroxyl radical formation. Biochemistry 31,
11606 –11613.
Jenner, A. M., and Timbrell, J. A. (1994). Influence of inducers and inhibitors
of cytochrome P450 on the hepatotoxicity of hydrazine in vivo. Arch.
Toxicol. 68, 349 –357.
Jones, D. P., Thor, H., Andersson, B., and Orrenius, S. (1978). Detoxification
reactions in isolated hepatocytes. Role of glutathione peroxidase, catalase,
and formaldehyde dehydrogenase in reactions relating to N-demethylation
by the cytochrome P-450 system. J. Biol. Chem. 253, 6031– 6037.
Aebi, H. (1984). Catalase in vitro. Methods Enzymol. 105, 121–126.
Kappus, H. (1987). A survey of chemicals inducing lipid peroxidation in
biological systems. Chem. Phys. Lipids 45, 105–115.
Anundi, I., Hogberg, J., and Stead, A. H. (1979). Glutathione depletion in
isolated hepatocytes: Its relation to lipid peroxidation and cell damage. Acta
Pharmacol. Toxicol. (Copenh.) 45, 45–51.
Kang, Y. J., Chen, Y., and Epstein, P. N. (1986). Suppression of doxorubicin
cardiotoxicity by overexpression of catalase in the heart of transgenic mice.
J. Biol. Chem. 271, 12610 –12616.
Blair, I. A., Mansilla Tinoco, R., Brodie, M. J., Clare, R. A., Dollery, C. T.,
Timbrell, J. A., and Beever, I. A. (1985). Plasma hydrazine concentrations
in man after isoniazid and hydralazine administration. Hum. Toxicol. 4,
195–202.
Kaneo, Y., Iguchi, S., Kubo, H., Iwagiri, N., and Matsuyama, K. (1984). Tissue
distribution of hydrazine and its metabolites in rats. J. Pharmacobiodyn. 7,
556 –562.
Bosan, W. S., Shank, R. C., MacEwen, J. D., Gaworski, C. L., and Newberne,
P. M. (1987). Methylation of DNA guanine during the course of induction
of liver cancer in hamsters by hydrazine or dimethylnitrosamine. Carcinogenesis 8, 439 – 444.
Cai, Y., Appelkvist, E. L., and DePierre, J. W. (1995). Hepatic oxidative stress
and related defenses during treatment of mice with acetylsalicylic acid and
other peroxisome proliferators. J. Biochem. Toxicol. 10, 87–94.
Kerai, M. D., and Timbrell, J. A. (1997). Effect of fructose on the biochemical
toxicity of hydrazine in isolated rat hepatocytes. Toxicology 120, 221–230.
Kenyon, S. H., Waterfield, C. J., Asker, D. S., Kudo, M., Moss, D. W., Bates,
T. E., Nicolaou, A., Gibbons, W. A., and Timbrell, J. A. (1999). Effect of
hydrazine upon vitamin B12-dependent methionine synthase activity and the
sulfur amino acid pathway in isolated rat hepatocytes. Biochem. Pharmacol.
57, 1311–1319.
Carlberg, I., and Mannervik, B. (1985). Glutathione reductase. Methods Enzymol. 113, 484 – 490.
Kleineke, J., Peters, H., and Soling, H.D. (1979). Inhibition of hepatic gluconeogenesis by phenethylhydrazine (phenelzine). Biochem. Pharmacol. 28,
1379 –1389.
Carmichael, J., DeGraff, W. G., Gazdar, A. F., Minna, J. D., and Mitchell, J. B.
(1987). Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing. Cancer Res. 47, 936 –942.
Kluck, R. M., Bossy-Wetzel, E., Green, D. R., and Newmeyer, D. D. (1997).
The release of cytochrome c from mitochondria: A primary site for Bcl-2
regulation of apoptosis. Science 275, 1132–1136.
Choudhary, G., and Hansen, H. (1998). Human health perspective on environmental exposure to hydrazines: a review. Chemosphere 37, 801– 843.
Loft, S., and Poulsen, H. E. (1999). Markers of oxidative damage to DNA:
Antioxidants and molecular damage. Methods Enzymol. 300, 166 –184.
Colvin, L. B. (1969). Metabolic fate of hydrazines and hydrazides. J. Pharm.
Sci. 58, 1433–1443.
Liu, X., Kim, C. N., Yang, J., Jemmerson, R., and Wang, X. (1996). Induction
of apoptotic program in cell-free extracts: Requirement for dATP and
cytochrome c. Cell 86, 147–157.
DelRaso, N. J., and Frazier, J. M. (1999). Effect of culture conditions prior to
432
HUSSAIN AND FRAZIER
Matsushita, M., Irino, T., Komoda, T., and Sakagishi, Y. (1993). Determination of proteins by a reverse biuret method combined with the copperbathocuproine chelate reaction. Clin. Chim. Acta 216, 103–111.
Moldeus, P., Hogberg, J., and Orrenius, S. (1978). Isolation and use of liver
cells. Methods Enzymol. 52, 60 –71.
Moloney, S. J., and Prough, R. A. (1983). Studies on the pathway of methane
formation from procarbazine, a 2-methylbenzylhydrazine derivative, by rat
liver microsomes. Arch. Biochem. Biophys. 221, 577–584.
Noda, A., Noda, H., Misaka, A., Sumimoto, H., and Tatsumi, K. (1988).
Hydrazine radical formation catalyzed by rat microsomal NADPH- cytochrome P-450 reductase. Biochem. Biophys. Res. Commun. 153, 256 –260.
Noda, A., Sendo, T., Ohno, K., Noda, H., and Goto, S. (1987). Metabolism and
cytotoxicity of hydrazine in isolated rat hepatocytes. Chem. Pharm. Bull.
(Tokyo) 35, 2538 –2544.
Ohkawa, H., Ohishi, N., and Yagi, K. (1979). Assay for lipid peroxides in
animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358.
Petersen, P., Bredahl, E., Lauritsen, O., and Laursen, T. (1970). Examination
of the liver in personnel working with liquid rocket propellant. Br. J. Ind.
Med. 27, 141–146.
Preece, N. E., Ghatineh, S., and Timbrell, J. A. (1990). Course of ATP
depletion in hydrazine hepatotoxicity. Arch. Toxicol. 64, 49 –53.
Preece, N. E., Ghatineh, S., and Timbrell, J. A. (1992). Studies on the
disposition and metabolism of hydrazine in rats in vivo. Hum. Exp. Toxicol.
11, 121–127.
Preece, N. E., and Timbrell, J. A. (1989). Investigation of lipid peroxidation
induced by hydrazine compounds in vivo in the rat. Pharmacol. Toxicol. 64,
282–285.
Roberge, A., Gosselin, C., and Charbonneau, R. (1971). Effect of hydrazine on
urea cycle enzymes in vitro and in vivo. Biochem. Pharmacol. 20, 2231–
2238.
Runge-Morris, M. A., Iacob, S., and Novak, R. F. (1988). Characterization of
hydrazine-stimulated proteolysis in human erythrocytes. Toxicol. Appl.
Pharmacol. 94, 414 – 426.
Seglen, P. O. (1976). Preparation of isolated rat liver cells. Methods Cell Biol.
3, 29 – 83.
Sendo, T., Noda, A., Ohno, K., Goto, S., and Noda, H. (1984). Hepatotoxicity
of hydrazine in isolated rat hepatocytes. Chem. Pharm. Bull. (Tokyo) 32,
795–796.
Sies, H. (1999). Glutathione and its role in cellular functions. Free Radical
Biol. Med. 27, 916 –921.
Sinha, B. K. (1987). Activation of hydrazine derivatives to free radicals in the
perfused rat liver: A spin-trapping study. Biochim. Biophys. Acta 924,
261–269.
Tietze, F. (1969). Enzymic method for quantitative determination of nanogram
amounts of total and oxidized glutathione: applications to mammalian blood
and other tissues. Anal. Biochem. 27, 502–522.
Timbrell, J. A., and Harland, S. J. (1979). Identification and quantitation of
hydrazine in the urine of patients treated with hydralazine. Clin. Pharmacol.
Ther. 26, 81– 88.
Wakabayashi, T., Teranishi, M. A., Karbowski, M., Nishizawa, Y., Usukura,
J., Kurono, C., and Soji, T. (2000). Functional aspects of megamitochondria
isolated from hydrazine- and ethanol-treated rat livers. Pathol. Int. 50,
20 –33.
Wald, N., Boreham, J., Doll, R., and Bonsall, J. (1984). Occupational exposure
to hydrazine and subsequent risk of cancer. Br. J. Ind. Med. 41, 31–34.
Wang, H., and Joseph, J. A. (1999). Quantitating cellular oxidative stress by
dicholorofluorescein assay using microplate reader. Free Radical Biol. Med.
27, 612– 616.
Willis, J. E. (1966). The substitution of 1-methylhydrazine for ammonia in the
glutamine synthetase system. Biochemistry 11, 3557–3563.
Wu, E. Y., Smith, M. T., Bellomo, G., and Di Monte, D. (1990). Relationships
between the mitochondrial transmembrane potential, ATP concentration,
and cytotoxicity in isolated rat hepatocytes. Arch. Biochem. Biophys. 282,
358 –362.
Yu, B. P. (1994). Cellular defenses against damage from reactive oxygen
species. Physiol. Rev. 74, 139 –162.