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thesis entitled

PLATINUM-INDUCED TOXICITY AND POTENTIAL
MODULATION BY CALCIUM TREATMENT:
A HISTOCHEMICAL STUDY

presented by

DANIEL J OSEPB MEARA

has been accepted towards fulfillment
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Date AUGUST 23, 1996

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PLATINUM-INDUCED TOXICITY AND POTENTIAL MODULATION BY
CALCIUM TREATMENT: A HISTOCHEMICAL STUDY

By

Daniel Joseph Meara

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Zoology

l 996

ABSTRACT

PLATINUM-INDUCED TOXICITY AND POTENTIAL MODULATION BY
CALCIUM TREATMENT: A HISTOCHEMICAL STUDY

By

Daniel Joseph Meara

Cisplatin (CDDP), Carboplatin (CBDCA) and Taxol (Paclitaxel) are potent anti-
neoplastic agents with associated toxicities that are their dose-limiting factors in clinical
oncology, especially Cisplatin-induced nephrotoxicity. In an attempt to elucidate their
mechanism(s) of toxicity, kidney and liver tissues from normal and drug treated wistar
rats and dogs were evaluated by analyzing changes in various dehydrogenase and
nonspecific lipase enzymes. Specifically, IDI-I, B-HBDH, GDH, MDH, SDI-I, LDH, and
G-6-PDH were studied using standard tetrazolium salt methods. Nonspecific lipases
were localized using the Tween method. Histochemically, Cisplatin treatment caused
morphological changes in the tissues as well as enzyme inhibition in all the enzymes
except G-6-PDH and nonspecific lipases. It is hypothesized that hydrolyzed Cisplatin
disrupts calcium homeostasis and binds to sulphydryl groups (-SH), resulting in inhibition
of ATP synthesis and modified membrane permeability. Further, lipases are activated
which strip enzymes from plasma membranes. However, carboplatin treatment results in
less toxicity as does Cisplatin plus taxol combination therapy. Supplemental treatments of

calcium (10 ml/kg of 1.3% CaClzlday in rats and 0.5 pg rocaltrol/day in dogs) seems to

protect against severe toxicity by protecting dehydrogenase enzymes and preserving

overall tissue morphology.

Dedicated to My Family

for their Love and Guidance

ACKNOWLEDGMENTS

Thanks to Dr. SK Aggarwal for being my mentor and friend. Also, thanks to Dr.
RA Fax and Dr. A Goya] for their invaluable interest and assistance as guidance
committee members. Thanks to Dr. JH Whallon for her help with the LSM and the
quantitative analyses. Thanks to Dr. R Nachreiner for his help with the ionized calcium
calculations. Thanks to Dr. Wang for his willingness to help and donate his time.
Thanks to the Zoology office staff: Chris Keyes, Tracey Bamer, Judy Pardee, Lisa Kraft
and Janice Mead for all their help. Lastly, thanks to Bristol Meyers and NIH for their

samples of Taxol, Cisplatin and Carboplatin.

TABLE OF CONTENTS
LIST OF TABLES ............................................................................................................ vi
LIST OF FIGURES ......................................................................................................... vii
INTRODUCTION ............................................................................................................. 1
MATERIALS AND METHODS ...................................................................................... 4
Animals ................................................................................................................. 4
Blood Collection and Analysis .............................................................................. 5
Tissue Collection ................................................................................................... 5
Histochemical Studies ........................................................................................... 5
Determination of Optimal pH and Incubation Time ................................. 5
Dehydrogenase (SDH, GDH, HBDH, MDH, IDH, LDH, G-6—PDH)
Localization ................................................................................... S
Nonspecific Lipase Localization ............................................................... 6
Photomicroscopy .................................................................................................. 6
Qualitative Analysis .............................................................................................. 6
Quantitative Analysis ............................................................................................ 6
RESULTS .......................................................................................................................... 9
Effects of Drug Treatments on Dehydrogenases and Nonspecific Lipases
in Rat Tissues ............................................................................................ 9
Comparison of Dehydrogenase Activity in Normal and Cisplatin Plus Calcium
Treated Dog Kidneys ............................................................................... 14
Control Slides for Dehydrogenases and Nonspecific Lipases After the Various
Treatments ............................................................................................... 15
Effects of Drug Treatments on Morphology of Rat Kidney and Liver Tissues
and Dog Kidney Tissues .......................................................................... 15
Optimal Enzyme Intensity and Localization at Variable pH Levels and Time
Schedules ................................................................................................. 16
Blood Collection and Analysis ............................................................................ 17
DISCUSSION .................................................................................................................. 44
BIBLIOGRAPHY ............................................................................................................ 53

LIST OF TABLES

Table 1. Experimental Design ........................................................................................... 8

Table 2. Average enzyme histochemical staining intensity for normal, treated, and
control tissues ................................................................................................................... 42

Table 3. Gray scale values indicating average enzymatic staining intensity for
normal, treated, and control tissues .................................................................................. 43

vi

LIST OF FIGURES

Figure 1. Light micrographs of cross-sections of kidney tissue showing the
localization of lactate dehydrogenase (LDH) ................................................................... 19

Figure 2. Light photomicrographs of kidney from normal and treated rat tissues showing
localization of malate dehydrogenase (MDH) .................................................................. 21

Figure 3. Light micrographs of normal and treated kidney tissues showing localization
of Glucose-6-phosphate dehydrogenase (G-6-PDH) ........................................................ 23

Figure 4. Bar graph showing enzymatic activity of dehydrogenase enzymes in normal,
treated, and control tissues. Intensity measurements are converted to percentages and
based on the normal tissue being equivalent to 100% staining intensity ......................... 25

Figure 5. Light photomicrographs of liver from normal and treated animal tissues
showing localization of malate dehydrogenase (MDH) ................................................... 27

Figure 6. Light micrographs of normal and treated liver tissues showing localization
of Glucose-6-phosphate dehydrogenase (G-6-PDH) ........................................................ 29

Figure 7. Light micrographs of normal and treated kidney tissues showing localization
of nonspecific lipase activity ............................................................................................ 31

Figure 8. Bar graph showing variable nonspecific lipase activity. Intensity measurements
are converted to percentages and based on the normal tissue being equivalent to 100%
staining intensity ............................................................................................................... 33

Figure 9. Light micrographs of normal and treated liver tissues showing localization
of nonspecific lipase activity ............................................................................................ 35

Figure 10. Light micrographs of normal and treated kidney tissues showing localization
of Isocitrate Dehydrogenase (IDH) and Glucose-6—phosphate dehydrogenase
(G-6-PDH) ....................................................................................................................... 37

Figure l 1. Graph showing optimal dehydrogenase intensity and localization at variable
pH and Time ..................................................................................................................... 39

vii

Figure 12. Graph showing ionized calcium levels during Cisplatin treatment cycle
adjusted for a pH of 7.4 .................................................................................................... 4]

viii

Figure 12. Graph showing ionized calcium levels during Cisplatin treatment cycle
adjusted for a pH of 7.4 .................................................................................................... 41

viii

INTRODUCTION

Cisplatin (cis-dichlorodiammineplatinum H), a heavy metal platinum coordination
complex, has received intense scrutiny as to its mechanism of action since its
serendipitous discovery by Barnett Rosenberg of Michigan State University in 1965
(Rosenberg et al., 1965). DNA denaturation is one of the accepted methods of its action
through its intrastrand and interstrand cross-links interfering with DNA replication and
transcription (Roberts and Pascoe, 1972; Rosenberg, 1975; Zwelling et al., 1979, 1981;
Fitchinger-Schepman et al., 1987; Reed et al., 1986). It has been shown that proteins
containing a high-mobility group (HMG) domain bind specifically to DNA modified by
Cisplatin, but not to unmodified DNA, and thus prevent excision repair (Huang et al.,
1994). Since clinical testing began around 1972, it has become clear that Cisplatin is
successful in reducing or even eliminating cancers of the head and neck (Jacobs et al.
1983), lung (Ruckdeschel et al., 1985), breast (Cox et al., 1989), ovary (W iltshaw et al.,
1985), bladder (Loehrer et al., 1990) and testicle (Williams and Einhom, 1980). Coupled
to its anti-tumor activity are many severe toxic side effects including peripheral
neuropathy (Kedar et al., 1978), ototoxicity (Piehl et al., 1974), myelosuppression (Talley
et al., 1973; Ozols et al., 1984), nausea and vomiting (Higby et al., 1974; Yagoda et al.,
1976), hypomagnesia (Schilsky and Anderson, 1979), hypocalcemia (Blachley and Hill,

1981), enzyme disruption (Aggarwal, 1993), hepatotoxicity (Cavalli et al., 1978) and

nephrotoxicity (Leonard et al., 1971). More specifically, it is the nephrotoxicity
associated with Cisplatin use that has become the dose-limiting factor in its use. It
manifests, pathologically, as renal tubular damage which results in elevation of the blood
urea nitrogen and serum creatinine levels (Hardaker et al., 1974). Renal damage from
Cisplatin treatment has been characterized as being similar to that of mercury and other
heavy metals which also result in renal tubular necrosis, degeneration, and interstitial
edema without glomerular changes (Madias and Harrington, 1978). This severe renal
toxicity caused Dr. J. Hill of the Wadley Institutes of Molecular Medicine, who was
responsible for the first report of CDDP’s clear anticancer activity in human patients, to
remark that, “Cis-platinum(II) diamminechloride appears to be too good a therapeutic
agent to abandon, yet too toxic for general use (Hill et al., 1974). It was later
demonstrated that hydrating patients markedly diminished kidney toxicity associated with
Cisplatin without a major loss of anticancer activity (Hayes et al., 1977). Slow infusion
rates have also been shown to ameliorate kidney toxicity (Merrin, 1976). Further,
antioxidants and thiol containing compounds, such as sodium thiosulfate, have become
part of the treatment regimen in an attempt to alleviate nephrotoxicity (Powis and Hacker,
1991). Despite these advances, nephrotoxicity is still a major concern and limitation in
Cisplatin chemotherapy treatment due to the extremely high uptake by the kidneys which
is approximately three times the uptake of any other organ (Wolf and Manaka, 1977).
Carboplatin (cis-diammine-1,1-cyclobutane dicarboxylate platinum II), is an

analogue of Cisplatin with a similarly proposed mechanism of action and comparable

effectiveness that has demonstrated reduced toxicity (Alberts et al., 1990). Consequently,
carboplatin has been suggested as an alternate to cisplatin use.

Also, cisplatin in combination with many other anticancer agents has proven to be
very effective (Woodman et al., 1973). Specifically, cisplatin plus taxol (paclitaxel) has
become increasingly prevalent in clinical treatment (Rowinsky et al., 1991). Combination
therapy is capable of producing response rates of up to 100% and is seemingly less toxic
than the either of the two anticancer compounds given alone (Donehower and Rowinsky,
1992).

Further, it has been shown that calcium supplementation can help protect enzyme
function and preserve overall organ function by minimizing the disruption of cellular
homeostasis initiated by cisplatin treatment (Aggarwal and Fadool, 1993). Thus, the
present study was undertaken to (1) characterize cisplatin-induced enzymatic changes in
rat kidney and liver tissues and dog kidney tissues, (2) compare carboplatin-induced
enzymatic changes to that of cisplatin treatment, (3) compare enzymatic changes
associated with taxol as well as enzymatic changes associated with combination therapy
(cisplatin plus taxol), (4) characterize structural and functional changes associated with

calcium supplementation and (5) correlate the various changes to the associated toxicities.

MATERIALS AND METHODS

MIME:

Wistar rats (Charles Rivers Laboratory, Wilmington, MA) weighing between 160-
200 g were used in the various experiments over a period of 17 months. Animals were
kept on a 12 hr light/l2 hr dark, cycle with access to laboratory animal food and water ad
libitum in accordance with the Guide for Care and Use of Laboratory Animals. Animals
received intraperitoneal (ip) injections of freshly prepared cisplatin, carboplatin, taxol,
cisplatin plus taxol, cisplatin plus 1.3% calcium chloride or calcium gluconate, or 0.85%
sodium chloride or 5% glucose in concentrations and dosages shown in Table 1.

Male dogs weighing 70—95 lbs were kept on a 12 hr light/12 hr dark cycle. The
dogs had free access to water and food in accordance with the Guide for Care and Use of
Laboratory Animals. Three dogs were twice infused with cisplatin ( 1.8 mg/kg) in
conjunction with saline solution and three received the vehicle alone. The dogs also

received 0.5 ug rocaltrol supplements (Roche Laboratories), by oral administration, for

five days prior to infusion and every day after infusion to maintain an elevated serum

calcium level.

Blood Collection and Analysis:

Blood samples were taken every day for the first week and then every third day
from then on and serum was tested for ionic calcium levels as well as blood urea nitrogen
(BUN) and creatinine levels.

Tissue Collection:

The rats in the various groups were anesthetized with C02 and killed by
decapitation at each sampling interval. The tissues (kidneys and livers) were quickly
excised and mounted on cryostubs in O.C.T. medium (Miles Laboratories, Elkhart, IN)
and frozen at -20°C until use. Sections (10 um) were cut using a cryostat microtome
(Miles Laboratories) for enzymatic analysis.

The dogs were given a lethal dose of sodium pentobarbital (325 mg/kg). The
tissues were quickly excised and mounted on cryostubs in O.C.T. medium (Miles
Laboratories) and frozen at -20°C. Sections (10 um) were cut for enzymatic analysis.
Histochemical Studies:

Determination of Optimal pH and Incubation Time. pH levels were varied from
7.0 to 7.6 while incubation time was kept constant. Incubation time was then varied from
20 minutes to 90 minutes while the pH was kept constant.

Dehydrogenase Localization. Frozen sections (10 pm) of normal, cisplatin,

cisplatin plus calcium, carboplatin, cisplatin plus taxol, and taxol treated tissues were
picked up on standard glass coverslips and allowed to dry at room temperature. Succinate

dehydrogenase (SDH), glutamate dehydrogenase (GDH), B-hydroxybutyrate

dehydrogenase (HBDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH),

lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6-PDH) were
then localized, histochemically, by the standard Nitro BT Method (Burstone, 1962).

The substrate or the coenzymes were omitted from the incubation media to serve
as controls. Tissue sections were also boiled in distilled water to denature the
endogenous enzymes and serve as a control.

Lipase Localization. Nonspecific lipase activity was localized by incubation of
tissue sections in a medium according to the methods of George and Ambadkar (1963)
and George and Iype (1960). Boiled tissue sections served as a control.
Photomicroscogy:

Slides were viewed and micrographs were prepared of transmitted images using a
Zeiss Photomicroscope II.

Qualitative analysis:

Staining intensity was based subjectively on an arbitrary scale from very intense
response (+++++) to intense response (++++) to moderate response (+++) to poor
response (++) to very poor response (+) to no response (-). Tissue sections were
randomly evaluated and the cortex and medulla were considered in the subjective
determination of enzyme localization response.

Cell size and general morphology, of the tissues, were evaluated by visual
observation and by light micrographs.

Qt_ian_tit_ative Analysis:
Approximately four transmitted images from each normal, cisplatin, carboplatin,

taxol, cisplatin plus taxol, and cisplatin plus calcium treated tissues, as well as control

slides, were examined by the Zeiss 10 Laser Scanning Confocal Microscope (LSM) and
quantitative analyses were made using the ‘histogram’ program within the LSM
computer. Random areas of the tissues were analyzed for staining intensity by the
computer and a representative histogram detailing ‘gray scale values’ was produced for
each enzyme. Statistical analysis was performed and gray scale values were then

converted to percentages based on normal staining being equivalent to 100% intensity.

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RESULTS

Effects of Drug Treatments on Dehydrogenases and Nonspecific Lipases in Rat Tissues:

Histochemically, of all the dehydrogenases studied, only the G-6-PDH
demonstrated an increase in its activity and distribution patterns after various treatments.
LDH and MDH resemble the activity and localization of the other dehydrogenases
studied: SDH, GDH, HBDH, and IDH. Thus, for the sake of brevity and simplicity, only
LDH, MDH, and G-6-PDH are discussed in detail for the rat kidneys and only MDH and
G-6-PDH are discussed for the rat livers.

Sections of normal kidney localized for the LDH and MDH showed dark blue
granulation with intense, diffuse staining throughout the cytoplasm of the tubules.
Specifically, in the normal kidney, staining intensity and localization was approximately
the same in both the cortical and medullar regions with the proximal and distal tubules
having equally pronounced enzymatic activity. Further, no staining was demonstrated
within the nuclei or glomeruli (Figures 1, 2, 4; Tables 2, 3). Similarly, sections of normal
liver localized for MDH showed dark and diffuse staining throughout the cytoplasm of
the hepatocytes. The nuclei of the hepatocytes were unstained (Figure 4, 5; Tables 2, 3).

In cisplatin treated kidneys, LDH and MDH enzymatic staining was decreased

compared to normal, showing indiscriminate and less intense granulation and gray

10

coloration throughout the cytoplasm of the cells. Further, enzymatic activity was
depressed in the medulla compared to the cortex and distal tubule staining was less than
that demonstrated within the proximal tubules (Figures 1, 2, 4; Tables 2, 3). Sections
from cisplatin treated livers stained for MDH demonstrated a significant decline in
enzymatic activity in the cytoplasm of the hepatocytes compared to normal (Figures 4, 5;
Tables 2, 3).

In the kidneys treated with cisplatin and calcium, LDH and MDH staining and
localization were maintained similar to normal. Staining was significant throughout the
cytoplasm of the cells of the tubules. The cortex and medulla were equally stained as
were the proximal and distal tubules. The nuclei and glomeruli showed no significant
staining (Figures 1, 2, 4; Tables 2, 3). The cisplatin plus calcium livers stained for MDH
again followed a similar pattern to the kidneys. Specifically, MDH staining and
localization was maintained similar to the normal tissues with staining being intense
throughout the cytoplasm of all the hepatocytes (Figures 4, 5; Tables 2, 3).

Sections of carboplatin treated kidneys localized for LDH and MDH also
demonstrated a decline in enzymatic activity, from normal levels, but less than cisplatin-
induced enzyme depression. Staining gradually decreased from the exterior of the cortex
to the medulla and the proximal and distal tubules declined equally (Figures 1, 2, 4;
Tables 2, 3). In livers treated with carboplatin, MDH enzymatic activity also declined in
a similar manner to that seen in the kidneys treated with carboplatin. MDH hepatocyte
staining was depressed compared to normal but less depressed than after cisplatin

treatment (Figures 4, 5; Tables 2, 3).

ll

Cisplatin plus taxol treated kidneys had LDH and MDH enzyme intensities greater
than cisplatin treatment alone but less than that seen in normal tissues. Medullar staining
was less than cortical and the proximal and distal tubules declined equally (Figures 1, 2,
4; Tables 2, 3). MDH localization in livers treated with cisplatin and taxol followed a
similar pattern. MDH hepatocyte staining was less than normal but greater than cisplatin
treatment alone (Figures 4, 5; Tables 2, 3).

LDH and MDH after taxol treatment demonstrated a minimal decline in activity
from normal. Cortical and medullar staining was approximately the same as was
proximal and distal tubule staining (Figures 1, 2, 4; Tables 2, 3). In livers similarly
treated with taxol, MDH activity also declined very little from normal levels. MDH
staining within the cytoplasm of the hepatocytes was diffuse and only slightly less intense
than that seen in the normal tissues (Figures 4, 5; Tables 2, 3).

Sections of normal kidney localized for G-6-PDH were similar to the other
dehydrogenases studied. Specifically, G-6-PDH staining was diffuse throughout the
cytoplasm of the tubules. Further, staining intensity and localization was approximately
the same in both the cortical and medullar regions with the proximal and distal tubules
having equally pronounced enzymatic activity. No significant staining was demonstrated
within the nuclei or glomeruli (Figure 3, 4; Tables 2, 3). Normal liver localized for 6-6-
PDH showed dark and diffuse staining throughout the cytoplasm of the hepatocytes
(Figures 3,4; Tables 2, 3).

However, in the kidneys and livers of the drug treated animals, the G-6-PDH

staining results were reciprocal to those seen with the other dehydrogenases studied,

12

except for cisplatin plus calcium. Specifically, cisplatin treated kidneys demonstrated
increased G-6-PDH staining throughout the tubules compared to normal. The cortical
and medullar regions had similarly increased G-6-PDH activity as did the proximal and
distal tubules (Figures 3, 4; Tables 2, 3). Liver cryo-sections stained for G-6-PDH also
demonstrated an increase in enzymatic activity throughout the cytoplasm of the
hepatocytes (Figures 4, 6; Tables 2, 3).

G-6-PDH activity after cisplatin plus calcium treatment was again maintained
close to normal levels. In the kidneys G-6-PDH activity closely approximated normal
staining with cortical and medullar regions as well as proximal and distal tubules being
equally stained (Figures 3, 4; Tables 2, 3). Similarly, cryo-sections of liver demonstrated
G-6-PDH hepatocyte staining similar to normal (Figures 4, 6; Tables 2, 3).

Carboplatin treatment also resulted in G-6-PDH enzyme elevation. G-6-PDH
elevation was consistent throughout the proximal and distal tubules of the cortex and
medulla. G-6-PDH staining after carboplatin was still less than after cisplatin treatment,
though both were elevated from normal (Figures 3, 4; Tables 2, 3). Similarly treated liver
demonstrated a slight increase in G-6-PDH enzymatic activity in the hepatocytes from
normal (Figures 4, 6; Tables 2, 3).

G-6-PDH activity after treatments of cisplatin plus taxol and taxol alone was
slightly greater than normal. The increase in G-6-PDH activity in the kidney cryo-
sections was approximately the same after both cisplatin plus taxol and taxol alone.
Specifically, distribution of G-6—PDH staining was equally elevated in the proximal and

distal tubules of the cortex and medulla (Figures 3, 4; Tables 2, 3). Similarly treated

l3

livers demonstrated similar G-6-PDH staining. Specifically, there was a slight increase in
G-6-PDH staining from normal after treatment with cisplatin plus taxol and taxol alone
with the increase being about the same after both treatments (Figures 4, 6; Tables 2, 3).

Nonspecific lipase activity followed the same pattern as that seen in G-6-PDH
activity. Normal kidney and livers localized for nonspecific lipase activity demonstrated
brown diffuse granulation throughout the tissues. In the kidneys, staining was similar in
the proximal and distal tubules of the cortex and medulla. No significant glomerular
staining was evident (Figures 7, 8; Tables 2, 3). Nonspecific lipase staining in normal
livers was similar to the normal kidneys in that there was diffuse granulation throughout
the cytoplasm of the hepatocytes (Figures 8, 9; Tables 2, 3).

Cisplatin treatment caused an elevation in lipase levels throughout the kidney
tubules compared to the normal tissues. The increase was consistent throughout the
cortex and medulla. No distinction between the proximal and distal tubules was evident
(Figures 7, 8; Tables 2, 3). Similarly, hepatocyte staining after cisplatin treatment was
also increased compared to normal (Figures 8, 9; Tables 2, 3).

Nonspecific lipase activity was closer to normal after cisplatin plus calcium
treatment in both the kidneys and livers. The proximal and distal tubules of the cortex
and medulla, in the kidneys, demonstrated equal staining that approximated normal levels
(Figures 7, 8; Tables 2, 3). Cytoplasmic staining in liver hepatocytes was also evenly
distributed and similar to normal (Figures 8, 9; Tables 2, 3).

Carboplatin treatment was similar to cisplatin treatment in that kidney cryo-

sections demonstrated an increase in activity compared to normal. However, lipase levels

14

increased less than after cisplatin treatment. Nonspecific lipase staining was consistent
throughout the cortex and medulla and no difference between proximal and distal tubules
was evident (Figures 7, 8; Tables 2, 3). Liver hepatocytes also showed a slight increase in
lipase activity compared to normal but the increase was less than that associated with
cisplatin treatment. Increased lipase activity in the liver cryo—sections after carboplatin
was consistent throughout the tissues with a slight increase in localization evident around
blood vessels of the hepatic lobules (Figures 8, 9; Tables 2, 3).

Cisplatin plus taxol treated tissues and taxol treated tissues both demonstrated
decreased lipase activity as compared to the normal tissues. Lipase activity after
combined treatment (cisplatin plus taxol) and taxol treatment was inconsistently spread
throughout the tissues. Though, staining was approximately equal in the cortex and
medulla and there was little distinction between the proximal or distal tubules (Figures 7,
8; Tables 2, 3). In the kidneys, taxol staining was slightly greater than seen in the
combined treatment. In the livers, cisplatin plus taxol combined treatment and individual
taxol treatment also demonstrated a decline in lipase activity throughout the hepatocytes
(Figures 8, 9; Tables 2, 3). However, combined cisplatin plus taxol staining was greater
than taxol treatment alone which is opposite to that seen in the kidney tissues where taxol
staining was slightly greater.

Compaiison of Dehydroge_nas‘e Activity in Normal_and Cisalatin Plus Calcium Treatgd
Dog Kidneys:
Sections of normal kidney localized for MDH and G—6-PDH demonstrated

moderate levels of blue granulation with diffuse staining throughout the cytoplasm of the

15

proximal and distal tubules. Staining was even and consistent throughout the cortex and
medulla (Figure 10). In the cisplatin plus calcium treated tissues, the MDH intensity and
localization was similar to normal tissues, though slightly depressed. The cyt0plasmic
staining of the tubules was even, precise, and comparable to the normal tissue
dehydrogenase staining. No differences were noticeable in the proximal and distal
tubules of the cortex and medulla. Further, enzyme intensity and localization was
approximately the same for G-6-PDH as for MDH and the other dehydrogenases studied
after cisplatin and cisplatin plus calcium treatments. (Figure 10).
Control Slides for Dehydrogenases and Nonsmcific Lipases After the Various
Treatments:

Control tissue sections, for all eight enzymes, demonstrated no appreciable
staining (Figures 4, 8; Tables 2, 3).
Effects of Drag Treatments on Momhology in Rat Kidnevand Liver Tissues and Dog

Kidney Tissues:

Normal kidney and liver tissues, stained for all the dehydrogenases and
nonspecific lipases studied, were morphologically more distinct due to improved clarity
of tissue structure. Specifically, normal tissues showed cylindrical-shaped cell
membranes and blood vessels as well as minimal intercellular tissue gaps (Figures 1-3, 7,
9, 10). However, cisplatin treated kidney and liver tissues demonstrated indistinct and
amorphous cell membranes and increased intercellular tissue space (Figures 1-3, 7, 9, 10).
Further, individual tubules in the kidneys, and hepatocytes in the livers, lacked

delineation from adjacent tubules and hepatocytes resulting in histologically indistinct

16

tissue structures as if the structures had merged together. Tissues treated with cisplatin
plus calcium demonstrated increased tissue organization and structure compared to the
morphology of the cisplatin treated tissues (Figures 1-3, 7, 9, 10). Cisplatin plus calcium
more approximated normal tissues which have clear boundaries between cells.
Carboplatin treated tissues were morphologically similar to normal tissues as carboplatin
treatment seemed to cause less morphological damage. Cisplatin plus taxol and taxol
treated tissues showed tissue morphology more similar to normal than the cisplatin

treated tissues (Figures 1-3, 7, 9, 10).

Optimal Enzyme Intensity and Localization at Variable pH LevelL and Time SChedl_1_l_§_§i

 

pH levels ranging from 7.0 to 7.2 demonstrated optimal dehydrogenase staining
over variable time schedules ranging from 20 minutes to 90 minutes (Figure 11).
Intensity was optimal in conjunction with localization. At pH levels above 7.2, staining
increased but was indiscriminate.

The average optimal incubation time for dehydrogenase staining was 45 minutes
(Figure 11). Staining for less than 45 minutes demonstrated incomplete staining in most
instances, however variability in incubation time occurred depending on the enzyme of
study. Incubation for more 60 minutes or more was also unsatisfactory as staining
became indiscriminate and occasionally excessive as the time increased.

A pH between 7.0 and 7.2 and an incubation time between 35 and 50 minutes was
the optimal dehydrogenase localization protocol. However, variation in optimal

enzymatic staining conditions occurred, but overall, staining anomalies, such as no

 

l7

reaction or ‘nothing dehydrogenase’ formation were prevented and staining was optimal

following the aforementioned optimal localization protocol (Figure 1 1).

Blood Collection and Analysis:

Serum calcium, blood urea nitrogen (BUN), and creatinine levels were maintained
at high normal physiological levels after calcium supplementation in cisplatin treated

dogs (Figure 12).

18

Figure 1. Light micrographs of cross-sections of rat kidney showing the localization of
lactate dehydrogenase (LDH). (A) Normal histological section showing strong presence
of LDH activity within the cytoplasm of the epithelium cells of the tubules (arrows). (B)
Cross-section of kidney tubules from cisplatin treated rat showing a remarkable decline in
LDH activity as compared to normal. Note the lack of staining in the glomeruli (g). (C)
LDH staining intensity after CDDP plus calcium treatment, demonstrating increased
enzyme activity, in the renal tubules, as compared to CDDP (arrow). (D) Cross-section
of carboplatin treated kidney showing slightly elevated LDH tubule staining compared to
cisplatin treated tissue though still less than normal. (E) Section of CDDP plus taxol
treated kidney demonstrating decreased LDH activity, in the cytoplasm, compared to
normal tissue, though greater than cisplatin treated kidneys. (F) Kidney tubules after
taxol treatment. Note that staining is comparable to the CDDP plus taxol treated tissue
section, though less than normal. Original magnifications: A-F: X 420. Bars = 40um.

 

20

Figure 2. Light photomicrographs from normal and treated rat kidneys showing
localization of malate dehydrogenase (MDH). (A) Normal histological section showing
intense MDH activity throughout the cytoplasm of the cells of the rat kidney tubules. (B)
Cross-section of cisplatin treated kidney showing a significant decline in MDH activity
throughout the tissue compared to normal. (C) Section of CDDP plus calcium treated
kidney demonstrating cortical tubules with preserved MDH activity similar to that of
normal, especially when compared to the cisplatin tissue section (arrow). (D) MDH
staining after carboplatin treatment is decreased as compared to normal but slightly
increased as compared to CDDP treatment. (B) Cross-section of CDDP plus taxol treated
kidney approximates the LDH activity of normal kidneys. (F) MDH activity in taxol
treated tissue section is comparable to normal. g, glomerulus. Original magnifications:
A-F: X 420. Bars = 40pm.

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22

Figure 3. Light micrographs of normal and treated rat kidneys showing localization of
glucose-6-phosphate dehydrogenase (G-6-PDH). (A) Normal histological section
showing G-6-PDH activity. (B) Cross-section of cisplatin treated kidney showing a
significant increase in G-6-PDH activity, compared to normal, in the cytoplasm of the
cells of the tubules (arrows). (C) G—6—PDH levels after CDDP plus calcium treatment
demonstrating enzymatic activity approximating the normal tissues. (D) Section of
carboplatin treated kidney showing a slight G-6-PDH increase from normal, though less
than with CDDP treatment. (E) Section of CDDP plus taxol treated kidney showing LDH
activity, in the tubules, comparable to normal. (F) G-6-PDH staining after taxol
treatment demonstrating enzymatic activity similar to normal. g, glomerulus. Original
magnifications: A-F: X 420. Bars = 40 um.

23

 

24

Figure 4. Bar graph showing comparative enzymatic activity of MDH and G-6—PDH in
normal and treated kidney and liver tissues. Note that MDH resembles SDH, GDH,
HBDH, IDH, and LDH activity. Note that 100% staining is the baseline and represents
normal tissue enzyme activity.

 

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26

Figure 5. Light photomicrographs from normal and treated rat livers showing localization
of malate dehydrogenase (MDH). (A) Normal liver section with hepatocytes with
unstained nuclei. Enzymatic staining is predominantly in the cytoplasm surrounding the
nuclei (arrow). (B) Cross-section after cisplatin treatment. Note the decline in enzymatic
activity in the cytoplasm of the hepatocytes. (C) Cross-section of cisplatin plus calcium
treated liver demonstrating MDH activity similar to normal tissues (arrows). (D) Section
of carboplatin treated liver showing decreased enzymatic staining throughout the
cytoplasm of the hepatocytes. Note that the enzymatic staining is greater than
demonstrated with cisplatin treatment. (B) MDH activity after cisplatin plus taxol
treatment demonstrating a slight decrease in enzymatic staining compared to normal and
(F) section of taxol treated liver showing slightly less staining than normal. bv, blood
vessel. Original magnifications: A-F: X 420. Bars = 40 um.

 

28

Figure 6. Light micrographs from normal and treated rat livers showing localization of
glucose-6-phosphate dehydrogenase (G-6-PDH). (A) Normal histological section
showing G-6-PDH activity. (B) Cross-section of (cisplatin treated liver showing a
significant increase in G-6-PDH activity in the cytoplasm of hepatocytes (arrows)
compared to normal. (C) G-6-PDH levels after CDDP plus calcium treatment
demonstrating enzymatic activity approximating the normal tissue section. (D)
Carboplatin treatment shows a slight G-6-PDH increase from normal, though less than
the increase in CDDP treated tissue. (E) CDDP plus taxol treatment demonstrates
enzymatic activity, in the hepatocytes, comparable to that of the normal tissues, though
slightly less. (F) G-6-PDH activity after taxol treatment demonstrating G-6-PDH
staining slightly less than normal. Original magnifications: A-F: X 420. Bars = 40 um.

29

 

30

Figure 7. Light micrographs of normal and treated animal kidneys showing localization
of nonspecific lipase activity. (A) Lipase distribution in normal kidney tubule section and
(B) cross-section after cisplatin treatment demonstrating a significant increase in lipase
activity compared to normal (arrows). (C) CDDP plus calcium treatment showing a
minimal decline in lipase localization, from that of normal and (D) carboplatin treatment
demonstrating a slight decline in lipase activity from that of the normal kidney tubules.
(E) Lipase reaction product after CDDP plus taxol treatment demonstrating a decrease in
enzyme activity compared to normal. (F) Section of taxol treated kidney showing slightly
more lipase activity than CDDP plus taxol treatment but less than normal. Original
magnifications: A-F: X 420. Bars = 40 um.

31

 

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32

Figure 8. Graph showing comparative nonspecific lipase activity for the various
treatments. Intensity measurements are converted to percentages (%) and based on
normal tissue being equivalent to 100% staining intensity.

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33

Enzyme Intensity (%)

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CDDP plus Calcium

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34

Figure 9. Light micrographs of normal and treated rat livers showing localization

of nonspecific lipase activity. (A) Lipase distribution in normal liver hepatocytes and (B)
cisplatin treatment demonstrating a significant increase in lipase activity (arrows). (C)
CDDP plus calcium treatment showing a minimal decline in lipase localization compared
to normal and (D) carboplatin treatment demonstrating a slight decline in lipase activity
from that of the normal liver hepatocytes. Note the cylindrical-shaped blood vessel (bv).
(E) Lipase reaction product after CDDP plus taxol treatment demonstrating a slight
decrease in enzyme activity as compared to normal. (F) Cross-section of taxol treated
liver showing less lipase activity than normal as well as less than in CDDP plus taxol
liver hepatocytes. Original magnifications: A-F: X 420. Bars = 40 um.

 

36

Figure 10. Light micrographs of normal and treated dog kidneys showing localization of
Isocitrate Dehydrogenase (IDH) and Glucose-6-phosphate dehydrogenase

(G-6-PDH). (A) Normal kidney cross-section with tubules stained for IDH activity. (B)
Cross-section of cisplatin plus calcium treated kidney demonstrating IDH activity slightly
less than that of the normal tissue sections. (C) Normal kidney cross-section
demonstrating G-6-PDH activity in cytoplasm of tubule cells. Note the unstained
glomerulus (g). (D) G-6-PDH activity after cisplatin plus calcium treatment showing
enzymatic levels similar to normal tissues (arrow). Original magnifications: A-D: X
260. Bars = 40 pm.

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38

Figure 11. Graph showing variable intensity levels at variable pH and time. Note that
optimal staining is considered to be at a pH of 7.1 and a time of 45 minutes and was
assigned a value representing 100% staining intensity.

39

Staining Intensity (%)

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Figure 12. Graph showing ionized calcium levels during cisplatin treatment cycle (21
days) adjusted for a normal physiological pH of 7.4.

41

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DISCUSSION

Platinum, a heavy metal, is of great interest for its wide spectrum of antitumor
activity (Rozencweig et al., 1977). Specifically, cisplatin, a platinum based compound, is
one of the most successful and widely used anticancer agents. However, it is associated
with many toxicities, the most severe being the nephrotoxicity (Leonard et al., 1971) as a
result of 90% of the drug being removed by renal mechanisms (Casper et al., 1979;
Jacobs et al., 1980). Cisplatin’s mechanism of action is not fully elucidated, however, it
is accepted that upon administration, cisplatin is absorbed by the blood supply and then
by nature of its lipophilicity, it enters cells by passive diffusion (Powis and Hacker,
1991). Inside the cell, under low chloride ion concentration, cisplatin hydrolyzes to form
aqua complexes (Jones et al., 1991; Mistry et al., 1989) which then migrate and bind to
proteins and DNA, exerting their effects by preventing replication and transcription and
repair mechanisms have been shown to reverse this process, giving additional support to
this explanation (Andrews and Howell, 1990). However, this does not explain the dose-
limiting nephrotoxicity associated with this anticancer agent. Presently, we have learned
to combat the toxicities associated with cisplatin treatment, through diuresis, slow

infusion rates, antioxidants and dose—scheduling but the cause of these toxicities is still

45

elusive and thus the absolute prevention of the toxicities remains impossible without
further clarification.

Ade et al.( 1996) have shown that organometals bind to a plasma membrane
receptor causing nonselective ionic channels to open, resulting in calcium influx from
extracellular stores. This extracellular calcium seems to be required for metal
stimulation. This influx of calcium then activates phospholipase C through stimulation of
a G protein. Concurrently, it is thought that this calcium influx also activates
phospholipase A2 which translocates from the cytoplasm to the membrane. This lipase
activation results in extensive membrane breakdown and the generation of toxic
metabolites, such as prostaglandins, through an increase in arachidonic acid levels as well
as activation of kinases, proteases, and endonucleases. This cascade leads to altered
membrane permeability, cell signaling, and cytoskeletal organization in addition to
compromised mitochondrial function and DNA fragmentation that ultimately results in
further activation of degradative processes, culminating in cell death (Orrenius and
Nicotera, 1994). Thus, it is hypothesized, that cisplatin may act similarly by initially
binding to the plasma membrane, causing a significant rise in cytosolic calcium levels
that initiates cell cytotoxicity which is further exacerbated by cisplatin’s diffusion inside
the cell where the parent compound hydrolyzes to its toxic form. Ultimately, it seems
that cisplatin’s toxicities result, primarily, from its disruption of intracellular calcium
homeostasis and from its ability to mimic calcium.

The initial event in cisplatin toxicity is likely the increase in intracellular calcium

levels from extracellular stores. This rise in intracellular Ca2+ disrupts normal signal

46

transduction, inhibits response to hormones and growth factors, compromises
mitochondrial function and cytoskeletal organization and activates degradative processes
(Orrenius and Nicotera, 1994). However, it possible that some of these toxicities may be
responsible for its antitumor activity. For instance, altered cytoskeletal organization
results in depolymerization of rnicrotubules and microfilaments and excessive nuclear
calcium levels results in DNA fragmentation (Orrenius and Nicotera, 1994).

Similar to ionized lead (Pb2+), it seems that cisplatin’s hydrolysis to cis-
[(NH3)2Pt(H20)2]2+ worsens the various cytotoxicities by inhibiting sulphydryl-containing
enzymes, specifically mitochondrial dehydrogenases, by mimicry of calcium (Ca2+). This
ability of cis-[(NH3)2Pt(H20)2]2+ to mimic endogenous Ca2+ is probably the most
important reason for the vast and severe toxicities associated with cisplatin treatment.
Specifically, earlier studies have shown that cisplatin binds to Ca2+-dependent sulphydryl
groups on proteins and results in platinum’s modification of protein chemical structure
and function and a decline in glutathione (GSH) levels (Zhang and Lindup, 1994). As
glutathione is depleted, the cell becomes more vulnerable to toxic compounds because
mitochondrial GSH seems to be essential in the regulation of inner mitochondrial
permeability and enzyme function by keeping sulphydryl groups (-SH) in the reduced
state. When the SH-groups, of enzymes, are not maintained in a reduced form, they
become inactivated (Bogin et al.. 1994). NADPH, which also helps to maintain reduced
sulphydryl groups, also declines with CDDP treatment, so consequently, the depletion of
glutathione (GSH) and NADPH seemingly results in the displacement of calcium, by

hydrolyzed cisplatin, and the inhibition of dehydrogenase enzymes, essential rate-

47

controlling enzymes in the tricarboxylic acid cycle. As a consequence of this biochemical
disruption of the TCA cycle, NADH stores are depleted and the uncoupling of oxidative
phosphorylation and the electron transport chain results in hydroxyl radical formation and
a consequent state of oxidative stress (Zhang and Lindup, 1993). These free radicals
attack polyunsaturated lipids and proteins and initiate lipid peroxidation. This process
becomes autocatalytic and causes severe damage to membrane integrity and progressive
disorder (Tyler, 1992). This lipid peroxidation, in conjunction with inhibition of
sulphydryl groups, leads to further loss of membrane integrity, potential, and permeability
which further exacerbates the resultant toxicity of cisplatin therapy (Zhang and Lindup,
1994). For instance, Na"-K+ ATPase and Cay-Mg2+ ATPase are affected by inhibition of
their essential sulphydryl groups (Orrenius et al., 1992) and this loss of mitochondrial
function and metabolism, thus creating a state of anoxia and additional ionic imbalance
(K+ efflux; Na+ and Cl- influx), cell swelling, and ultimately cell lysis (Batzer and
Aggarwal, 1986). Further, this binding of heavy metals, such as platinum, to -SH groups
also induces a rapid release of calcium from intracellular storage organelles (Abramson
and Salama, 1989) and ATPase is unable to uptake calcium and restore equilibrium
(Binet and Volfin, 1977) thus worsening the disruption of calcium homeostasis initiated
by the earlier influx of extracellular calcium.

Concurrent with hydrolyzed cisplatin’s binding to Cay-dependent sulphydryl
containing enzymes, is its competitive binding at calmodulin binding sites. This
molecular mimicry activates the calmodulin complex in an unregulated manner (Jarve

and Aggarwal, l996) which may further inactivate Cab-sensitive dehydrogenases by

48

acting in conjunction with the elevated cytoplasmic calcium, which itself acts as a second
messenger. This induction of hyperrnetabolism is a cellular means of trying to
compensate for increased energy needs as result of mitochondrial damage but this also
leads to the additional activation of unregulated Ca2+—dependent degradative enzymes
such as phospholipases (Tyler, 1992) which in turn may alter cytoskeletal organization
and membrane permeability. Alkaline phosphatase, Na+lK+-ATPase, and Cay/Mg“-
ATPase have been shown to be stripped from their membranes and released into the urine
(Aggarwal, 1993) as an indirect result of cisplatin treatment, further altering ionic
homeostasis. Bound dehydrogenases may also be stripped from their mitochondrial
membrane by the hyperactivated lipases and nonmembrane bound, soluble
dehydrogenases, may be lost by damage to the tubules which results in leaky epithelium
allowing unregulated movement into and out of cells. Both events further impair ATP
formation.

However, glucose-6-phosphate dehydrogenase is seemingly enhanced like lipase
activity due to cisplatin treatment. It seems that elevation of glucose-6-phosphate
dehydrogenase activity occurs because of its unique and critical function in NADPH
generation and maintenance of reduced sulphydryl groups as part of the pentose
phosphate pathway (Raven, I994). Furthermore, if the pentose phosphate pathway is
carried through to completion, there is the degradation of glucose to glyceraldehyde-B-
phosphate, which can be further oxidized to produce ATP. Glucose-6-phosphate

dehydrogenase catalyzes the conversion of B-D-glucose-o-phosphate to 6-

phosphoglucono-l ,5-lactone and NADP is reduced in the process (Raven, 1994). Thus,

49

glucose-6-phosphate dehydrogenase has a dual function in being the rate determining step
in the pentose phosphate pathway. Its lack of essential sulphydryl groups seems to limit
its own inhibition and allows for increased activity that may be a means for cells to adapt
to the effects of cisplatin by inducing systems, such as glucose-6-phosphate
dehydrogenase, that produce NADPH to compensate for the decrease of free sulphydryl
groups (Bogin et a1, 1994). This appears to be a cytoprotective mechanism against
cisplatin toxicity.

The initial rise in intracellular calcium is transient but it dulls the cells responses
to hormones and other trophic stimuli and eventually, cisplatin’s binding to proteins and
competition with calcium for calcium binding sites, induces persistent hypocalcemia
(Brenner, 1996). Hypocalcemia continues the disruption of calcium homeostasis and
thus, Ca2+-dependent enzymes such as the dehydrogenases, are inhibited, enhancing the
cisplatin-induced cytotoxicities. However, calcium supplementation seems to modulate
cisplatin-induced toxicities. Modified membrane permeability and regulation, due to
treatment, seems to allow exogenous calcium to enter the cells and reestablish calcium
homeostasis by competing with hydrolyzed cisplatin for binding sites on Can-dependent
enzymes, allowing for normal enzymatic activity and reduced organ toxicity. We have
demonstrated this in the kidney where calcium supplementation maintained normal serum
calcium levels as well normal blood urea nitrogen (BUN) and creatinine levels.

Carboplatin (CBDCA), an analogue of cisplatin, has been touted as an effective
alternative to cisplatin treatment. Depending on the type of cancer, carboplatin has been

shown to be equivalent or slightly superior to cisplatin in randomized trials (Alberts et al.,

50

1990). However, there are also many toxicities also associated with its use.
Thrombocytopenia is dose-limiting but leukopenia and anemia also occur but to a lesser
degree (Calvert et al., 1982). Interestingly, CBDCA nephrotoxicity is less severe and
studies have shown that calcium levels are only rarely decreased with its use (Canetta, et
al., 1985). In this study, dehydrogenase enzyme levels showed less decline in activity and
tissue morphology showed less disruption than that demonstrated with cisplatin
treatment, suggesting decreased nephrotoxicity associated with CBDCA. This seems to
be a result of carboplatin’s chemical structure and its consequent slow hydrolysis, which
results in the need for clinical dosages 10 times that of cisplatin (Harstruck et al., 1989)
for similar therapeutic success. Although lipase activity is still slightly activated by
treatment, it is probable that carboplatin’s slow hydrolysis limits its binding to
dehydrogenase enzymes and allows endogenous protective mechanisms, such as GSH, to
combat any potential toxicity associated with its use.

Another alternative to cisplatin anticancer treatment is cisplatin plus taxol
combined therapy. Taxol (paclitaxel) is a diterpene plant product with no heavy metal
constituents (Wani et al., 1971). Further, it is highly active in numerous preclinical tumor
models (Rose et al., 1992). But again, taxol treatment is associated with a number of
toxicities such as hypersensitivity (Weiss et al., 1990), alopecia (Wiernik et al., I987) and
emesis (Brown et al., 1991). Taxol’s combination with cisplatin has proven extremely
effective against breast cancer with success rates approaching 100% (Donehower and
Rowinsky 1992) and nephrotoxicity that is limited. Dehydrogenase inhibition is minimal

and lipase activity is approximately normal. There are four possible explanations for this

51

minimal nephrotoxicity. First, taxol binds extensively to proteins but lacks the structural
design allowing it to mimic calcium and thus, its effects on dehydrogenases are limited.
Second, taxol is eliminated by hepatic conversion, by the cytochrome P450 system,
(Rowinsky and Donehower, 1991) which minimizes its nephrotoxicity due to limited
renal exposure. Third, in combination therapy, the maximum tolerated dosage (MTD) of
the two chemotherapeutic agents is approximately 50% of their individual dosages.
Consequently, this decrease in cisplatin levels leads to less dehydrogenase inhibition, less
mitochondrial disruption and thus, less overall cytotoxicity. Lastly, it has recently been
shown that taxol and cisplatin combination therapy is schedule dependent. Addition or
even synergism occurs when taxol is given approximately 24 hours prior to cisplatin
treatment but when cisplatin is given prior to taxol or if they are administered at the same
time, strong antagonistic interactions are observed (Vanhoefer et al., 1995).
Consequently, all of these factors are probably responsible for the appreciable decline in
nephrotoxicity; the most significant being the schedule dependency of combination
therapy as in this study. Nonetheless, clinical success of cisplatin plus taxol combination
therapy is a viable alternative to cisplatin treatment alone.

Hepatotoxicity from the various treatments, especially cisplatin, is minimal
because of the liver’s ability to maintain calcium homeostasis through sequestration of
calcium in the sarcoplasmic reticulum and the abundant mitochondria in the liver. Also,
the liver contains large glutathione (GSH) stores which can protect against hydrolyzed
cisplatin (Bellomo et al., 1983). Further, accumulation of the compounds in the liver is

less than that in the kidney (Wolf and Manaka, 1977). However, the slight toxicities

52

(enzyme and morphological alterations) that do occur are seemingly modulated by
calcium supplementation.

In conclusion, from this study it seems clear that cisplatin’s disruption of calcium
homeostasis and metabolism to reactive metabolites, that mimic endogenous elements,
initiates primary events such as lipid peroxidation, changes in thiol status and enzyme
inhibition. These events damage the cells of the kidney tissues and secondary events
occur. Such events include: changes in membrane structure, mitochondrial damage,
inhibition of mitochondrial function, depletion of ATP and other cofactors, and further
changes in Ca2+ levels. These result in final manifestations or tertiary events such as
apoptosis and tissue necrosis (T imbrell, 1991). Simply-stated, cisplatin’s disruption of
calcium homeostasis and inactivation of dehydrogenase enzymes, in conjunction with
lipase activity, results in the considerable decrease in dehydrogenase enzymatic activity
which is directly responsible for ATP disruption and indirectly responsible for most of the
associated toxicities that result from this disruption. Thus, it seems that elevated calcium
levels, via calcium supplementation, may act as another means of cyto-protection, along
with endogenous glucose-6-phosphate dehydrogenase, by competing for binding sites
with hydrolyzed cisplatin and thus preventing cisplatin from initiating a cascade of

cytotoxic events.

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