Introduction

Top Abstract Introduction Methods Results Discussion References

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 Single strand DNA breaks occur on the DNA strand on which guanine has been replaced by thioguanine (Pan and Nelson, 1990) probably due to blockage of strand extension because of its poor ability to act as a substrate for polymerase and DNA ligase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).

View larger version (35K): [in this window] [in a new window]   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).

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.

	Material and Methods

Top Abstract Introduction Methods Results Discussion References

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.

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).

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).

	Results

Top Abstract Introduction Methods Results Discussion References

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.

View larger version (116K): [in this window] [in a new window]   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.)

View larger version (86K): [in this window] [in a new window]   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.)

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.

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 × 10⁶ mol) i.p. in 48 hrs intervals (Wu and Melton, 1993).

6

	Discussion

Top Abstract Introduction Methods Results Discussion References

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.

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).

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).