Department of Molecular and Cellular Toxicology, Harvard School of
Public Health, Boston, Massachusetts (J.A., R.H.S.) and
Etex
Corporation, Cambridge, Massachusetts (M.E.P.G.)
6-Thioguanine (6TG) a cytostatic antimetabolite is currently used to
treat patients with cancer, in particular leukemias. However, one
drawback of such use is the development of 6TG resistance. Hypoxanthine-guanine phosphoribosyl transferase (Hprt) plays a crucial
role in the bioactivation of 6TG. Loss of Hprt has been associated with
the resistance of leukemias to 6TG chemotherapy, however, nothing has
been known about the effect of Hprt status on tissue specific toxicity
of 6TG in vivo. We determined the effect of Hprt status on
the tissue-specific toxicity of 6TG in vivo in transgenic
Hprt-deficient mice. The approximate lethal dose for Hprt-deficient
mice was 23-fold higher than for the wild-type. Serum biochemical
analyses of 6TG-treated wild-type mice showed elevated serum enzyme
levels characteristic of liver damage whereas the levels in
Hprt-deficient 6TG-treated mice were within normal physiological
limits. Histopathological examination of tissues from wild-type and
from Hprt-deficient mice showed contrasting spectrums of microscopic
lesions. Wild-type mice had loss of hematopoietic cells from bone
marrow starting at the lowest dose of 25 mg/kg 6TG whereas
Hprt-deficient mice had normal bone marrow and spleen even at doses of
720 mg/kg 6TG. Wild-type mice also experienced severe loss of
epithelial cells from the gastrointestinal tract starting at 50 mg/kg;
however, the gastrointestinal tract of Hprt 
/ mice
remained unaffected. Wild-type livers revealed atrophy and necrosis at
doses of 25 mg/kg 6TG although Hprt / livers displayed
no effect until 507 mg/kg. In this study we show that Hprt-deficient
mice had 6TG-resistant bone marrow and there are several other factors
contributing to 6TG resistance in patients. Because variations among
people exist in terms of their 6TG sensitivity, determining 6TG
sensitivity of lymphocytes prior to 6TG chemotherapy and restricting
treatment to 6TG-sensitive patients may improve the efficacy.
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Introduction |
Cytotoxic
drug resistance is a major obstacle to successful chemotherapy in
cancer patients. 6TG is effective as leukemia treatment agent as well
as immunosupressant (Calabresi and Parks, 1985
; Loo and Nelson, 1982).
It has been noted that virtually all major current protocols for
"average" and "low risk" ALL include as a core component of
continuing chemotherapy daily doses of 6-mercaptopurine (6MP), an
analog of 6TG that is metabolized in the same way (Lennard and
Lilleyman, 1989). 6TG is first converted to 6TGMP by Hprt in the purine
salvage pathway (fig. 1, (Calabresi and
Parks, 1985)). The biological activity of this product is several-fold.
First, 6TGMP works as a pseudofeedback inhibitor of
glutamine-5-phosphoribosylpyrophosphate amidotransferase and blocks
purine biosynthesis. Second, 6TGMP inhibits IMP dehydrogenase and thus
purine interconversion. The net consequence of this activity is a block
of the synthesis and utilization of purine nucleotides (Calabresi and
Parks, 1985). Third, 6TGMP, after conversion to the tri-phosphate form
is incorporated into either DNA or RNA (LePage, 1963; Ling et
al., 1992; Pan and Nelson, 1990). Single strand DNA breaks occur
on the DNA strand on which guanine has been replaced by thioguanine
(Pan and Nelson, 1990) probably due to blockage of strand extension
because of its poor ability to act as a substrate for polymerase and
DNA ligase (Ling et al., 1992). 6TG is eliminated from the
body mostly in the form of inactive metabolites which include 6TX, 6TUA
and 6MeTG [fig. 1; (Elion, 1967; LePage and Whitecar, 1971)]. The
distribution of these metabolites seems to be different in mice and man
(Elion, 1967; Elion et al., 1963; LePage and Whitecar,
1971).
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Fig. 1.
Mechanism of action and metabolic fate of
6-thioguanine. 6TG is converted to its monophosphate (6TGMP) by Hprt.
This active metabolite interferes with de novo synthesis of purines by
pseudofeedback inhibition of phosphoribosyl pyrophosphate (PRPP)
amidotransferase and by blocking of IMP dehydrogenase. Furthermore,
6TGMP is phosphorylated to triphosphate (6TGTP) and incorporated into
DNA and RNA. The biodegradation of 6TG in the mouse consists mostly of
deamination and oxidation to inactive metabolites 6-thioxanthine (6TX)
and 6-thiouric acid, respectively. Small portion of 6TG also excreted in the form of 6-methyl-thioguanine (6MeTG).
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Several laboratory and clinical observations suggest that Hprt
deficiency causes cellular resistance to 6TG. For example, cells from
Lesch-Nyhan syndrome patients lack Hprt and are resistant to 6TG
(Dempsey et al., 1983; Yamanaka et al., 1985).
Most chemically induced mutant cells that are resistant to 6TG show
significantly reduced Hprt activity (Sato et al., 1972),
reviews (Caskey and Kruh, 1979; Siminovitch, 1976). Many leukemia
patients treated with 6MP develop 6MP resistance; hence the drug fails
to maintain remission of the disease, e.g., Brockman, 1974).
Among 15 analyzed cases of 6MP-resistant leukemias one was due to
complete loss of Hprt activity (Davidson and Winter, 1964). Additional
mechanisms of such 6TG-resistance include lower affinity of Hprt for
the ribose-phosphate donor PRPP, increased degradation of 6MP,
decreased incorporation of the analog into polynucleotides, and failure of the analog to enter the cells (Brockman, 1974). Prevention of 6TG
toxic effects can be achieved by administration of the purines adenine
or hypoxanthine in leukemia cells (Hashimoto et al., 1990)
or by adenosine in vitro and in vivo (Epstein and
Preisler, 1984). This protective effect has been explained as a
decrease of 6TG bioactivation by competition for PRPP (Hashimoto
et al., 1990), or by its depletion (Epstein and Preisler,
1984).
Although the correlation between Hprt deficiency and 6TG resistance is
established very well in cultured cells (Sato et al., 1972),
reviews (Caskey and Kruh, 1979; Siminovitch, 1976) there exist little
or no data that address in vivo levels of Hprt activity and
whole animal or tissue-specific 6TG toxicity. Hprt might be involved in
toxic side effects of 6TG therapy and relative or absolute Hprt
deficiency could play an important role in the development of 6TG
resistant tumors.
We determined the effect of the Hprt status on 6TG toxicity in
vivo. We used transgenic Hprt-deficient mice that completely lack
Hprt enzymatic activity (Hooper et al., 1987; Koller
et al., 1989). Although Hprt deficiency in humans causes a
behavioral and neurological disorder called Lesch-Nyhan syndrome (Lesh
and Nyhan, 1964; Seegmiller et al., 1967), Hprt-deficient
mice are clinically and behaviorally within normal limits (Hooper
et al., 1987; Koller et al., 1989). This may be
explained by increased purine de novo synthesis in the mice
(Jinnah et al., 1993) and by alternative salvage of purines
by Aprt (Wu and Melton, 1993).
As a quantitative parameter of the acute 6TG toxicity in Hprt-deficient
and Hprt wild-type mice we determined the approximate lethal
dose that corresponds to the LD50 ± 30% for most
chemicals (Deichmann and LeBlanc, 1943). Furthermore, we performed
histopathological and serum biochemical analysis of treated wild-type
and Hprt-deficient animals. These studies determined the effect of Hprt
status on the spectrum of target organ specificity of 6TG toxicity.
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Material and Methods |
Animals and drug treatment.
Transgenic Hprt-deficient female
mice were obtained from Dr. B. Koller (University of North Carolina,
Chapel Hill, NC). These mice have a 129/J genetic background and carry
a deletion of exons 1 and 2 of the Hprt gene (Hooper
et al., 1987). Control animals, wild-type 129/J mice, were
obtained from the Jackson Laboratory (Bar Harbor, ME). The mice were
kept in SPF conditions, provided with standard diet and water ad
libitum. Animal care and experimental procedures were carried out
in agreement with institutional guidelines.
6-Thioguanine (2-amino-6-merkaptopurine) was purchased from Sigma
Chemical Co. (St. Louis, MO). The compound was suspended in distilled
water and sonicated for 10 min before each i.p. injection. 9-ethyladenine (Sigma Chemical Co., St. Louis, MO) was dissolved in
sterile saline and stored at 4°C until i.p. application.
Acute toxicity, approximate lethal dose.
The approximate
lethal dose of 6TG after single i.p. injections was determined
(Deichmann and LeBlanc, 1943). In summary, the animals were treated
with single doses of 6TG i.p. at concentrations starting at 100 mg/kg
that increased by 50% for each consecutive dose. Control mice were
treated with the same volume of sterile distilled water. Because the
LD50 is known for wild-type mice this protocol was modified
for doses below 100 mg/kg, and concentrations of only 25 and 50 mg/kg
were used to save animals. The lowest dose at which the first animal
died was the approximate lethal dose. This dose corresponds to the
LD50 ± 30% for most chemicals (Deichmann and LeBlanc,
1943; Deichmann and Mergard, 1948). This protocol significantly reduces
the number of experimental animals, limits unnecessary suffering and
complies with current Guideline of Animal Studies (IARC, 1993).
Two sets of 8-month-old female animals were used for all acute toxicity
experiments. The 6TG suspension was injected i.p. The controls were
injected with sterile water. The health status of the animals was
observed twice daily and body weight was measured daily for 14 subsequential days. The dead or killed moribund mice were immediately
necropsied, and the organs were stored in 10% buffered neutral
formalin until further analysis. All survivors were killed with
pentobarbital (300 mg/kg, i.p.) 14 days after 6TG administration and
necropsies were performed. Necropsies consisted of a gross examination
of all external surfaces and orifices and all internal organs.
Histopathological examination and serum biochemical analysis of
serum.
The organs were prepared as paraffin-embedded tissue glass
slides stained with hematoxylin and eosin and evaluated according to
the NTP (National Toxicology Program of NIEHS) standards. A complete
cross-section of each organ, when possible, was evaluated (liver,
spleen, gastrointestinal tract, femoral bone marrow, mandibular and
mesenteric lymph nodes, kidney, brain, uterus and ovaries, lungs and
heart). For liver, two cross-sections, one of each of the two largest
liver lobes were examined. For kidneys, an entire cross-section (left
longitudinal, right transverse) were evaluated. Lungs had two
cross-sections (one of each of the two largest lobes). The entire
sections on the slides (all fields) were evaluated under blinded
conditions for lesions and scored (graded) on a subjective basis
compared to control animals. The grades were as follows: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, and - = no
pathological changes. The preparation and evaluation of slides used the
NTP criteria and terminology (Chhabra et al., 1990).
Serum biochemistry parameters analyzed included BUN, AST, ALT, CK and
AP. The analyses were performed at Tufts University Veterinary Medical
Diagnosis Laboratory with the chemical analyzer Hitachi 747. Blood was
collected from the posterior vena cava of Hprt deficient mice that were
either treated with water, 25,500 or 720 mg/kg 6TG i.p. and wildtype
mice administered 25 mg/kg 6TG.
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Results |
Acute toxicity, approximate lethal dose.
Hprt-deficient and
wild-type mice tolerated a single i.p. injection of 6TG without
immediate toxic symptoms or distress. The approximate lethal dose of
6TG for wild-type mice was 50 mg/kg (table
1). Hprt deficiency caused a dramatic
increase in resistance to 6TG. The approximate lethal dose for
Hprt / animals was 1148 mg/kg, which represents a
23-fold increase over the wild-type strain. Lethal doses in both Hprt
+/+ and Hprt / strains caused the same symptoms including loss of
body weight, decreased activity progressing to lethargy and coma.
Sublethal doses in Hprt / animals caused no visible
effects or changes of body weight. However, at the 25-mg/kg sublethal
dose for the wild type mice a 7% decrease in body weight was found.
Histopathological examination.
Complete histopathological
analysis of wild-type mice and of Hprt-deficient mice was performed for
all doses used for table 2 up to 720 and
2571 mg/kg, respectively. Some control Hprt-deficient mice had liver
lesions characterized by moderate to marked hepatocellular fatty change
typified by large distinct cytoplasmic vacuoles and centrilobular
hypertrophy. These lesions did not show any correlation with 6TG dose
and did not occur in wild-type control or 6TG-treated mice until lethal
doses of 225 mg/kg for the fatty change and 150 mg/kg for centrilobular
hypertrophy. Therefore, these lesions may be the consequence of Hprt
deficiency.
Lesions seen in wild-type and Hprt mutant mice differed
strikingly (table 2) for the same doses of 6TG. Starting at 6TG doses of 25 mg/kg Hprt wild-type mice had liver lesions such as
necrosis of scattered individual hepatocytes and atrophy of hepatocytes typified by decreased cell size. At higher doses, we observed centrilobular hypertrophy or increased relative size of centrilobular compared to other hepatocytes. In contrast, Hprt-deficient mice did not
show necrosis until doses of 507 mg/kg or more, and even at these
higher doses necrosis was less severe. In contrast to untreated
controls and treated wild-type mice, all Hprt-deficient mice treated
with sublethal doses of 6TG (100-720 mg/kg) showed slight
extramedullary hematopoiesis in the liver, which receded at lethal
doses.
Wild-type mice even at the lowest dose of 25 mg/kg 6TG experienced
depletion and necrosis of hematopoietic tissues, specifically the
femoral bone marrow. In addition, cells of the marrow and splenic red
pulp were absent or necrotic (table 2; fig.
2). Mandibular and mesenteric lymph nodes
had depletions in cell numbers. In contrast, Hprt-deficient mice
administered doses up to 720 mg/kg 6TG had normal to hypercellular bone
marrow, spleen and lymph nodes (table 2; fig. 2). In fact, all
Hprt-deficient mice treated with sublethal doses of 6TG from 50 to 720 mg/kg showed hypercellular bone marrow whereas none of the wild-type
mice did.
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Fig. 2.
Microscopic lesions in the bone marrow of wildtype
(Hprt +/+) mice treated with 100 mg/kg 6TG (A) compared to
Hprt deficient (Hprt /) mice administered 507 mg/kg of
6TG (B). A, Bone marrow of wildtype (Hprt +/+) mice treated
with 100 mg/kg 6TG. Uniform, small round to discoid anuclear cells are
mature erythrocytes (open arrowhead). They may have entered into vacant
spaces and are not usually prominent in normal bone marrow. Vessels and
fibrous connective stroma (solid arrowhead) are prominent due to
decreased number of normal marrow elements. The very few remaining bone marrow hematopoietic precursor cells (open arrow) have indistinct or
disrupted cytoplasm and/or pyknotic or fragmented nuclei diagnostic of
necrosis. B, Bone marrow of Hprt-deficient (Hprt /) mice administered 507 mg/kg of 6TG. The bone marrow from a treated Hprt-deficient mouse is within normal limits. Usual numbers and ratios
of hematopoietic cells are present (open arrow, megakaryocytes; solid
arrow, neutrophil cell lineage). Compared to wildtype, erythrocytes are
not prominent. (Magnification 100×, staining hematoxilin and eosin.)
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Wild-type mice showed atrophy and/or necrosis of gastrointestinal
epithelium (stomach, small and large intestines) such as loss of cells
(ulcer/erosion) and decreased thickness of cell layers and height of
villi and individual epithelial cells starting at doses of 50 mg/kg 6TG
(table 2, fig. 3). In contrast,
Hprt-deficient mice had normal to very minimal changes of
gastrointestinal tract epithelium.
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Fig. 3.
A, Intestinal epithelium of wild-type
(Hprt +/+) treated with 100 mg/kg 6TG. Representative
section of intestine from a wild-type mouse with epithelial surface
(solid arrow), submucosa (solid arrowhead) and muscle and serosal
layers (open arrow). The epithelium has marked atrophy and necrosis
characterized by decreased size and number of cells (open arrowhead).
B, Intestinal epithelium of Hprt-deficient (Hprt /) mice
administered 720 mg/kg 6TG. Intestine from Hprt-deficient mouse has
epithelium within normal limits. The epithelial surface (solid arrow)
has normal size and numbers of cells as do the submucosa (solid
arrowhead) and muscle layers (open arrow). (Magnification 100×,
staining hematoxilin and eosin.)
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Kidneys and other tissues examined in wild-type mice were within normal
limits. In contrast, 6TG-treated Hprt-deficient mice at sublethal doses
(150-720 mg/kg) had kidney lesions including scattered dilated renal
tubules, focal interstitial inflammation, focal tubule basophilia and
some glomerulopathy characterized by increased numbers of cells (table
2).
Hprt deficient mice administered with lethal doses of 6TG (1148 mg/kg
or more) had lesions of liver, spleen and bone marrow similar to those
observed in the wild-type mice treated with 25 mg/kg or more (table 2).
The kidney lesions in Hprt-deficient mice decreased at toxic doses
(table 2).
Serum biochemical analysis.
Hprt wild-type mice
were treated with the sublethal dose of 25 mg/kg 6TG and
Hprt mutant mice were treated with the sublethal doses of
25,500 and 750 mg/kg 6TG. Serum samples taken 14 days after treatment
were evaluated for the levels of BUN, AST, ALT, CK and AP. Elevated BUN
is associated with dehydration and/or renal insufficiency. Elevated
activities of ALT and AST are characteristic for liver damage,
particularly necrosis, cirrhosis and/or hepatitis and also muscle
trauma or myocardial infarction or myositis. Elevated AP is associated
mostly with increased bone marrow metabolism and also with
hepatocellular damage during hepatitis. CK is predominantly located in
muscles and therefore its increased activities are consequence of
muscular trauma, myocardial infarction or myopathic disorders.
Administration of a sublethal dose of 25 mg/kg 6TG resulted in
clinically significant elevated levels of AST, and ALT (table 3A) in Hprt wild-type mice
suggesting hepatocellular possibly necrotic damage in those animals.
Levels of CK and AP did not increase suggesting that 6TG does not cause
muscular damage or bone disorders. There was about a doubling of BUN
levels, slightly above the physiological range. Because we did not
detect histological evidence for renal toxicity of 6TG in wild-type
mice, these slightly elevated levels of BUN could be the result of
dehydration caused by impaired gastrointestinal epithelia or due to
decreased fluid intake. In contrast, Hprt-deficient mice, even after
sublethal doses of 720 mg/kg, showed no clinically significant changes
of the serum biochemical parameters (table 3B).
Effect of the Aprt inhibitor 9-ethyl adenine on 6TG toxicity in
Hprt-deficient mice.
Aprt catalyzes phosphorylation of adenine
to its monophosphate. Although this reaction is adenine specific
(Blakley, 1986) it might be possible that Aprt also phosphorylates
guanine (in our case 6TG) in the Hprt-deficient background in the
presence of massive amounts of 6TG. Thus, it might be possible that
inhibition of Aprt enzymatic activity actually decreases toxicity of
6TG in Hprt-deficient mice. It has been shown that Aprt activity is inhibited in mice by injection of 9-ethyl adenine (2.5 × 10
6 mol) i.p. in 48 hrs intervals (Wu and Melton, 1993).
To determine whether Aprt may activate 6-TG in Hprt-deficient mice we
treated such mice with five injections of 9-ethyladenine (2.5 × 106 mol) in 48-hr intervals before 6TG application. The
control groups received 9-ethyladenine or saline, respectively. Three
mice were used per group. 9-ethyladenine and saline-pretreated animals
received injections with the lethal dose of 6TG (1000 mg/kg). One group of 9-ethyladenine-treated animals served as a control for possible side
effects of Aprt inhibition. The treatment with 9-ethyladenine alone did
not result in any apparent toxic symptoms. All 6TG-treated animals
including 9-ethyladenine as well as saline pretreated mice showed the
same symptoms of 6TG toxicity that resulted in coma and death 5 days
after the 6TG injection. Thus, 9-ethyladenine pretreatment did not
protect Hprt-deficient animals against 6TG toxicity suggesting that
Aprt may not be responsible for the remaining toxicity of 6TG in
Hprt-deficient mice.
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Discussion |
6TG has been used for the treatment of leukemias and as an
immunosuppressive agent for several decades. However, to our knowledge little or nothing is known about the effect of Hprt status on the
tissue-specific toxicity of 6TG in animals. Thus, we determined the
effect of Hprt status on the tissue specific toxicity of 6TG in
vivo in transgenic Hprt-deficient mice. The approximate lethal dose for Hprt-deficient mice was 23-fold higher than for the wild-type. Serum biochemical analyses of 6TG-treated wild-type mice showed elevated serum enzyme levels characteristic of liver damage whereas the
levels in Hprt-deficient 6TG-treated mice were within normal physiological limits. Histopathological examination of tissues from
wild-type and from Hprt-deficient mice showed contrasting spectra of
microscopic lesions. Wild-type mice had loss of hematopoietic cells
from bone marrow starting at the lowest dose of 25 mg/kg 6TG whereas
Hprt-deficient mice had normal bone marrow and spleen even at doses of
720 mg/kg 6TG. Wild-type mice also experienced severe loss of
epithelial cells from the gastrointestinal tract starting at 50 mg/kg;
however, the gastrointestinal tract of Hprt / mice
remained unaffected. Wild-type livers revealed atrophy and necrosis at
doses of 25 mg/kg 6TG although Hprt / livers displayed
no effect until 507 mg/kg.
Acute toxicity of 6TG.
As a quantitative parameter of 6TG
toxicity we determined the approximate lethal dose that corresponds to
the LD50 ± 30% for most chemicals (Deichmann and LeBlanc,
1943). The approximate lethal dose in wild-type 129/J mice was 50 mg/kg. The previously published LD50 value of 90 mg/kg was
obtained in mice (Philips et al., 1954). Considering that
different mouse strains were used our approximate lethal dose
corresponds well with that value.
Acute toxicity of 6TG is delayed so that mice treated with the lowest
lethal dose survived for 8 to 13 days, and even mice treated with 10 times the approximate lethal dose still survived for 4 days after
administration (table 1). Delayed toxicity of 6TG has been found
previously and has been proposed to result from agranulocytosis or
thrombocytopenia and to resemble the toxic effects of ionizing
radiation (Philips et al., 1954). It is likely that 6TG has
to be metabolized and that the active 6TG metabolites cause damage in
proliferating or metabolizing tissue. This may have caused animals
death due to liver, hematopoietic and/or gastrointestinal failure.
These effects are seen with most of the other purine analog
chemotherapeutic agents (Philips et al., 1954).
Tissue specific toxicities of 6TG.
Tissues from
Hprt wild-type and Hprt mutant mice given a range
of 6TG doses had different spectra of microscopic lesions. It has been
previously shown that 6TG toxicity at fatal doses in wild-type mice is
predominantly limited to the bone marrow (Philips et al.,
1954). Our data show, that besides anticipated loss of hematopoietic
cells, the 6TG treated wild-type mice also had loss of gastrointestinal
epithelial cells and liver necrosis. Livers from 6TG-treated
Hprt wild-type mice had atrophy, centrilobular hypertrophy
and some liver cell necrosis. These lesions may be due to local effects
of 6TG metabolites produced in the hepatocytes. Observed liver damage
correlated with serum biochemical analysis by increased levels of ASP
and ALT (table 3A). It seems interesting that in rats and dogs damage
to the bone marrow seems to be the primary cause of death after 6TG
administration and that only slight if any damage to the liver is found
(Philips et al., 1956). However, in man in agreement with
our data myelosuppression is the primary complication of 6TG therapy
and orointestinal mucositis and hepatitis were frequent secondary side
effects (Bleyer, 1985).
Hprt deficiency in the transgenic mice caused marked protection for
hematopoietic tissues and intestinal epithelium against 6TG toxicity.
Hprt / mice given sublethal doses (720 mg/kg or less) of
6TG had normal bone marrow and gastrointestinal tract epithelium.
However, after lethal doses (1148 mg/kg or higher) the animals
displayed bone marrow lesions similar to treated wild-type mice (table
2). One possible explanation for this observation is activation of 6TG
by an alternate metabolic pathway with lower specificity for 6TG.
Although, Aprt is considered specific for adenine (Blakley, 1986) we
cannot exclude that high doses of 6TG in these animals might compete
with adenine for enzymatic conversion to 6TGMP. Cells deficient in Hprt
activity frequently exhibit elevated Aprt activity (Brockman, 1974;
Davidson and Winter, 1964). Thus, inhibition of Aprt enzymatic activity
might decrease toxicity of 6TG in Hprt-deficient mice. It has been
shown that Aprt activity is inhibited in mice by injection of
9-ethyladenine (Wu and Melton, 1993). Hprt-deficient mice do not show
any symptoms of the human Lesch-Nyhan syndrome; however, Aprt
inhibition in Hprt-deficient mice resulted in clinical manifestation of
Lesch-Nyhan syndrome. However, our experiment indicates that
9-ethyladenine pretreatment did not protect Hprt-deficient animals
against 6TG toxicity suggesting that Aprt is not responsible for the
remaining toxicity of 6TG in Hprt-deficient mice. Although it has been
shown that 9-ethyladenine inhibits Aprt activity in the brains of
Hprt-deficient mice, no data on the inhibition of Aprt activity in the
bone marrow and liver exist. Thus, as alternative explanation for our
results we cannot completely rule out that Aprt was not inhibited by
9-ethyladenine in the target organs of 6-TG toxicity.
Renal lesions including evidence of tubule and glomerular damage seen
only in Hprt / mice (table 2) may be associated with excretion of large amounts of 6TG catabolites such as 6-thiourea in the
kidneys. Administration of adenine, purine or 2-chloroadenine causes
the "adenine kidney" characterized by precipitation of crystals and
induction of lesions (Philips et al., 1954). However, such
crystals were not visible in our study or after administration of 6MP
in mice or man (Philips et al., 1954). It is possible that the 6TG catabolites do not form visible crystals but still occur and
cause irritation and lesions. Renal lesions were mainly developed at
sublethal 6TG doses of 150 to 720 mg/kg in Hprt-deficient mice. Wild-type mice did not survive these doses. Hprt-deficient mice at
lethal doses (1148 mg/kg or more) also had diminished renal lesions
(table 2) indicating that biodegradation, excretion and/or time might
be required for changes seen at sublethal doses. However, the 6TG
catabolic products have not been characterized in Hprt-deficient animals. Thus, different catabolic products might be formed in Hprt-deficient mice that are more toxic to kidneys.
Implications for cancer treatment.
There is some suggestive
evidence that Hprt levels might be important for the efficacy of 6TG
cancer treatment. Among 120 children with ALL, patients with lower
incorporation of 6TG into 6TG nucleotides show a significant higher
risk of relapse than patients with higher incorporation (Lennard and
Lilleyman, 1989). Furthermore, among 83 children with untreated ALL,
low Hprt activity is correlated with a poorer prognosis (Pieters
et al., 1992). Similarly, among 44 children with ALL, the
probability of continuous complete remission was significantly lower in
patients with 6TG-resistant cells (Pieters et al., 1991).
Furthermore, patients with untreated chronic lymphocytic leukemia had
significantly lower Hprt activities than control subjects and the Hprt
activities were quite widely dispersed (Rambotti and Davis, 1981).
However, other studies did not find a correlation between 6TG or 6MP
resistance and HPRT activity (Davidson and Winter, 1964; Pieters
et al., 1992). The underlying reason may be that a variety
of other factors may contribute to drug resistance including lower
affinity of Hprt for the ribose-phosphate donor
5
-phosphoribosyl-1-pyrophosphate, increased degradation of the drug,
decreased incorporation of the analog into polynucleotides, and failure
of the analog to enter the cells (Brockman, 1974).
However, there is quite some interindividual variation in 6TG
resistance of cells of phenotypically normal individuals (Yamanaka et al., 1985). There is also considerable interindividual
variability of the frequency of mutations giving rise to 6TG resistance
(Davies et al., 1992) and normal, nontransformed cells from
patients with cancer prone diseases such as Werners syndrome (Fukuchi
et al., 1990). Ataxia telangiectasia (Cole and Arlett,
1994), Bloom's syndrome (Vijayalaxmi et al., 1983) and
xeroderma pigmentosum (Cole et al., 1992) show a
significantly elevated frequency of mutations to 6TG resistance. Thus,
interindividual differences in 6TG sensitivity of lymphocytes (Lennard
and Lilleyman, 1989) and of the frequency of mutations causing 6TG
resistance may be responsible in part for the variability of the
efficacy observed for 6TG chemotherapy.
Bone marrow cell hyperproliferation of surviving cells is commonly a
result of toxicity to bone marrow cells (Jutter and To, 1992). Thus, if
an increased fraction of 6TG-resistant bone marrow cells exist, killing
of the 6TG-sensitive cells by 6TG might select for such 6TG-resistant
cells. To avoid this, the sensitivity of lymphocytes to 6TG could be
determined before and during administration of 6TG by lymphocyte
cloning or other methods (Dempsey et al., 1983; Veerman and
Pieters, 1990; Yamanaka et al., 1985).
The general approach of using transgenic animals with specific defects
in drug metabolism pathways can contribute to the understanding of the
mechanisms of toxicity and action of drugs. This could ultimately lead
to improvement of drug design and treatment regimes.
The authors thank Dr. B. Koller for donating
Hprt-deficient mice, the members of the Schiestl laboratory
for discussions and comments on the manuscript and Kristie Wetzel for
providing histology support services.
6MeTG, 6-methyl-thioguanine;
6MP, 6-mercaptopurine, 6TG, 6-thioguanine;
6TGMP, 6-thioguanine
monophosphate;
6TGTP, 6-thioguanine triphosphate;
6TUA, 6-thiouric
acid;
6TX, 6-thioxanthine;
ALL, acute lymphoblastic leukemia;
Hprt, hypoxanthine-guanine phosphoribosyl transferase;
IMP, inosine
monophosphate;
LD50, median lethal dose;
PRPP, phosphoribosyl pyrophosphate;
BUN, blood urea nitrogen;
AST, aspartate
amino transferase;
ALT, alanine amino transferase;
CK, creatine kinase;
AP, alkaline phosphatase;
Aprt, adenine phosphoribosyltransferase.
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Abstract
Introduction
